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2.  Azithromycin increases in vitro fibronectin production through interactions between macrophages and fibroblasts stimulated with Pseudomonas aeruginosa 
Objectives
Chronic azithromycin therapy has been associated with improved clinical outcomes in patients with cystic fibrosis (CF) who are chronically infected with Pseudomonas aeruginosa. We have previously demonstrated that azithromycin polarizes macrophages towards an alternatively activated phenotype, thereby blunting inflammation associated with infection. Because this phenotype is pro-fibrotic, it is important to evaluate azithromycin's consequential effects upon fibroblast function and extracellular matrix (ECM) protein production.
Methods
We co-cultured macrophages and fibroblasts together and stimulated them by adding P. aeruginosa or lipopolysaccharide to assess the ability of azithromycin to alter the macrophage phenotype, along with the impact exerted upon the production of fibronectin and other effectors that govern tissue remodelling, including transforming growth factor β (TGFβ), matrix metalloproteinase-9 (MMP-9) and arginase. We supported these studies by evaluating the impact of azithromycin treatment on these proteins in a mouse model of P. aeruginosa infection.
Results
Azithromycin increased arginase expression in vitro, as well as the activation of latent TGFβ, consistent with polarization to the alternative macrophage phenotype. While the drug increased fibronectin concentrations after stimulation in vitro, secretion of the ECM-degrading enzyme MMP-9 was also increased. Neutralization of active TGFβ resulted in the ablation of azithromycin's ability to increase fibronectin concentrations, but did not alter its ability to increase MMP-9 expression. In P. aeruginosa-infected mice, azithromycin significantly decreased MMP-9 and fibronectin concentrations in the alveolar space compared with non-treated, infected controls.
Conclusions
Our results suggest that azithromycin's effect on MMP-9 is regulated independently of TGFβ activity. Additionally, the beneficial effects of azithromycin may be partially due to effects on homeostasis in which ECM-degrading mediators like MMP-9 are up-regulated early after infection. This may impact the damaging effects of inflammation that lead to fibrosis in this patient population.
doi:10.1093/jac/dks476
PMCID: PMC3594493  PMID: 23248239
P. aeruginosa; bacterial infections; lung inflammation
3.  Fluconazole versus an echinocandin for Candida glabrata fungaemia: a retrospective cohort study 
Objectives
We studied whether fluconazole or echinocandin treatment of Candida glabrata fungaemia results in superior outcomes.
Methods
A multicentre, retrospective study was performed with 224 adult patients who received ≥5 days of therapy with either fluconazole or an echinocandin as their first antifungal treatment after collection of a blood culture that grew C. glabrata. The primary outcome was day 14 complete response.
Results
Patients in the echinocandin group were generally more ill, both at baseline and at the time of the index culture. Day 14 complete response was obtained in 58/127 (46%) and 50/97 (52%) of the fluconazole and echinocandin patients, respectively (P = 0.383). Logistic regression found intensive care unit admission to be associated with failure [OR 0.456 (0.217–0.957), P = 0.038] and echinocandin therapy to be associated with day 14 complete response [OR 2.305 (1.124–4.727), P = 0.023]. Twenty-eight day survival was similar between the fluconazole and echinocandin groups and logistic regression did not reveal antifungal therapy choice to be independently predictive of mortality. For patients treated with fluconazole, a dose : MIC ratio >12.5 (when compared with a ratio ≤12.5) was associated with a significantly higher day 14 complete response [4/20 (20%) ≤12.5 versus 50/102 (49%) >12.5, P = 0.025].
Conclusions
Severity of illness and choice of antifungal predict response in patients with C. glabrata fungaemia. Antifungal choice, however, does not influence mortality. In addition, new CLSI C. glabrata fluconazole susceptibility breakpoints are predictive of response when fluconazole is dosed appropriately.
doi:10.1093/jac/dks482
PMCID: PMC3594495  PMID: 23212115
pharmacodynamics; breakpoints; ICU
4.  Antitubercular pharmacodynamics of phenothiazines 
Objectives
Phenothiazines have been shown to exhibit in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb) and multidrug-resistant Mtb. They are predicted to target the genetically validated respiratory chain component type II NADH:quinone oxidoreductase (Ndh). Using a set of compounds containing the phenothiazine pharmacophore, we have (i) investigated whether chemical validation data support the molecular target and (ii) evaluated pharmacophore tractability for further drug development.
Methods
Recombinant Mtb Ndh was generated and its functionality confirmed by steady-state kinetics. Pharmacodynamic profiling of the phenothiazines, including antitubercular efficacy in aerobic and O2-limited conditions, time–kill assays and isobole analyses against first-line antituberculars, was performed. Potential mitochondrial toxicity was assessed in a modified HepG2 cell-line assay and against bovine cytochrome bc1.
Results
Steady-state kinetic analyses revealed a substrate preference for coenzyme Q2 and an inability to utilize NADPH. A positive correlation between recombinant Ndh inhibition and kill of aerobically cultured Mtb was observed, whilst enhanced potency was demonstrated in a hypoxic model. Time–kill studies revealed the phenothiazines to be bactericidal whilst isobolograms exposed antagonism with isoniazid, indicative of intracellular NADH/NAD+ couple perturbation. At therapeutic levels, phenothiazine-mediated toxicity was appreciable; however, specific mitochondrial targeting was excluded.
Conclusions
Data generated support the hypothesis that Ndh is the molecular target of phenothiazines. The favourable pharmacodynamic properties of the phenothiazines are consistent with a target product profile that includes activity against dormant/persistent bacilli, rapid bactericidal activity and activity against drug-resistant Mtb by a previously unexploited mode of action. These properties warrant further medicinal chemistry to improve potency and safety.
doi:10.1093/jac/dks483
PMCID: PMC3594496  PMID: 23228936
tuberculosis; thioridazine; Ndh; latency; toxicity
5.  Unexpected N-acetylation of capreomycin by mycobacterial Eis enzymes 
Objectives
The enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Eis_Mtb), a regio-versatile N-acetyltransferase active towards many aminoglycosides (AGs), confers resistance to kanamycin A in some cases of extensively drug-resistant tuberculosis (XDR-TB). We assessed the activity of Eis_Mtb and of its homologue from Mycobacterium smegmatis (Eis_Msm) against a panel of anti-tuberculosis (TB) drugs and lysine-containing compounds.
Methods and results
Both enzymes acetylated capreomycin and some lysine-containing compounds, but not other non-AG non-lysine-containing drugs tested. Modelling studies predicted the site of modification on capreomycin to be one of the two primary amines in its β-lysine side chain. Using Eis_Mtb, we established via nuclear magnetic resonance (NMR) spectroscopy that acetylation of capreomycin occurs on the ɛ-amine of the β-lysine side chain. Using Msm, we also demonstrated for the first time to our knowledge that acetylation of capreomycin results in deactivation of the drug.
Conclusions
Eis is a unique acetyltransferase capable of inactivating the anti-TB drug capreomycin, AGs and other lysine-containing compounds.
doi:10.1093/jac/dks497
PMCID: PMC3594498  PMID: 23233486
antibiotic resistance; anti-tuberculosis drugs; mechanisms of resistance
6.  The membrane as a target for controlling hypervirulent Clostridium difficile infections 
Objectives
The stationary phase of Clostridium difficile, which is primarily responsible for diarrhoeal symptoms, is refractory to antibiotic killing. We investigated whether disrupting the functions of the clostridial membrane is an approach to control C. difficile infections by promptly removing growing and non-growing cells.
Methods
The bactericidal activities of various membrane-active agents were determined against C. difficile logarithmic-phase and stationary-phase cultures and compared with known antibiotics. Their effects on the synthesis of ATP, toxins A/B and sporulation were also determined. The effect of rodent caecal contents on anti-difficile activities was examined using two reutericyclin lead compounds, clofazimine, daptomycin and other comparator antibiotics.
Results
Most membrane-active agents and partially daptomycin showed concentration-dependent killing of both logarithmic-phase and stationary-phase cultures. The exposure of cells to compounds at their MBC resulted in a rapid loss of viability with concomitant reductions in cellular ATP, toxins A/B and spore numbers. With the exception of nisin, these effects were not due to membrane pore formation. Interestingly, the activity of the proton ionophore nigericin significantly increased as the growth of C. difficile decreased, suggesting the importance of the proton gradient to the survival of non-growing cells. The activities of the lipophilic antimicrobials reutericyclins and clofazimine were reduced by caecal contents.
Conclusions
These findings indicate that C. difficile is uniquely susceptible to killing by molecules affecting its membrane function and bioenergetics, indicating that the clostridial membrane is a novel antimicrobial target for agents to alleviate the burden of C. difficile infections.
doi:10.1093/jac/dks493
PMCID: PMC3695663  PMID: 23264511
bactericidal; drug tolerant; non-growing cells
7.  Effect of pravastatin and fosinopril on recurrent urinary tract infections 
Objectives
Recurrent urinary tract infections (UTIs) are a problem affecting both women and men. Animal experiments and in vitro studies indicate that statins might prevent recurrent UTIs. We assessed the effects of pravastatin on UTI antibiotic prescribing among adults.
Methods
A post hoc analysis was conducted with data from PREVEND IT, a trial among participants randomized to receive pravastatin, fosinopril or placebo in a 2 × 2 factorial design over 4 years. Trial data were linked to the pharmacy prescription database IADB.nl. The primary outcome was the number of prescriptions with a nitrofuran derivate, a sulphonamide or trimethoprim as a proxy for UTI antibiotic prescribing. Generalized estimating equations were used to estimate the effect on the number of UTI antibiotic prescriptions. Cox regression was used to determine the effect on first and second (recurrent) UTI antibiotic prescriptions.
Results
Of the 864 trial participants, 655 were eligible for analysis. During an average follow-up of 3.8 years, 112 (17%) participants received at least one UTI antibiotic prescription. Intention-to-treat analyses showed that pravastatin was associated with a reduced total number of UTI antibiotic prescriptions (relative risk, 0.43; 95% CI, 0.21–0.88) and occurrence of second UTI antibiotic prescriptions [hazard ratio (HR), 0.25; 95% CI, 0.08–0.77]. No significant effect on occurrence of first UTI antibiotic prescriptions was found (HR, 0.83; 95% CI, 0.57–1.20). Fosinopril was associated with an increased occurrence of first UTI antibiotic prescriptions (HR, 1.82; 95% CI, 1.16–2.88). Combination therapy with fosinopril and pravastatin did not significantly influence the number of UTI antibiotic prescriptions.
Conclusions
This study suggests that pravastatin can reduce the occurrence of recurrent UTIs. Larger studies among patients with recurrent UTIs are warranted.
doi:10.1093/jac/dks419
PMCID: PMC3566666  PMID: 23111852
cystitis; statin; bacterial invasion
8.  Improving existing tools for Mycobacterium xenopi treatment: assessment of drug combinations and characterization of mouse models of infection and chemotherapy 
Background
Mycobacterium xenopi is a common agent of non-tuberculous mycobacterial lung diseases in Europe. However, an optimal treatment regimen for M. xenopi infection has not yet been established. Appropriate in vitro and in vivo model systems are needed for characterization of the activity of potential drugs and drug combinations against M. xenopi.
Methods
We utilized three experimental platforms to analyse the anti-M. xenopi activity of single and combination drug regimens. First, we determined the bacteriostatic and bactericidal activities of drugs alone and in combination in vitro. Second, we used serum from treated mice to evaluate drug activities ex vivo. Third, we analysed M. xenopi growth in four strains of mice (BALB/c, C57BL/6, beige and athymic nude) and developed a mouse model of chemotherapy for this infection.
Results
Two-drug combinations of ethambutol with rifampicin, rifapentine or moxifloxacin, and of clarithromycin with moxifloxacin were bactericidal in vitro, and the combination of ethambutol and rifampicin with either clarithromycin or moxifloxacin showed significant bactericidal activity ex vivo. Nude mice were the most susceptible strain to M. xenopi infection, and in this model amikacin-containing regimens were the most effective against M. xenopi. No difference in activity was found between regimens containing clarithromycin and moxifloxacin in vivo.
Conclusion
The ethambutol/rifampicin combination with clarithromycin or moxifloxacin had significant bactericidal activity against M. xenopi. The nude mouse, being highly susceptible to M. xenopi, can be utilized for in vivo chemotherapy studies for this infection.
doi:10.1093/jac/dks421
PMCID: PMC3566667  PMID: 23129730
drug susceptibility testing; clarithromycin; ethambutol; nude mouse
9.  Pharmacokinetics and efficacy of a vaginally administered maraviroc gel in rhesus macaques 
Objectives
To investigate the pharmacokinetics (PK) of maraviroc, a CCR5-targeted HIV-1 entry inhibitor, in rhesus macaques following vaginal administration of various maraviroc-loaded aqueous hydroxyethylcellulose (HEC) gels, and to correlate the PK data with efficacy in a single high-dose vaginal SHIV-162P3 challenge model.
Methods
Maraviroc concentrations in vaginal fluid (Weck-Cel® sponge), vaginal tissue (punch biopsy) and plasma were assessed over 72 h following single-dose vaginal application of various maraviroc-loaded HEC gels. The range of maraviroc gel concentrations was sufficiently broad (0.003%–3.3% w/w) that test gels included both fully solubilized and predominantly dispersed formulations. The efficacy of the HEC gels against a single high-dose vaginal SHIV-162P3 challenge was also measured, and correlated with the PK concentrations.
Results
Maraviroc concentrations in vaginal fluid (range 104–107 ng/mL), vaginal tissue (100–1200 ng/g) and plasma (<102 ng/mL) were highly dependent on maraviroc gel loading, irrespective of the form of the maraviroc component within the gel (solubilized versus dispersed). Fluid and plasma concentrations were generally highest 0.5 or 2 h after gel application, before declining steadily through to 72 h. Maraviroc concentrations in the various biological compartments correlated strongly with the extent of protection against vaginal SHIV-162P3 challenge. Complete protection was achieved with a 3.3% w/w maraviroc gel.
Conclusions
A high degree of correlation between PK and efficacy was observed. Based on the data obtained with the 3.3% w/w maraviroc gel, maintenance of vaginal fluid and tissue levels in the order of 107 ng/mL and 103 ng/g, respectively, are required for complete protection with this compound.
doi:10.1093/jac/dks422
PMCID: PMC3566668  PMID: 23111849
HIV; vaginal microbicide; drug delivery
10.  Antibacterial activity of the human host defence peptide LL-37 and selected synthetic cationic lipids against bacteria associated with oral and upper respiratory tract infections 
Objectives
We aim to develop antibacterial peptide mimics resistant to protease degradation, with broad-spectrum activity at sites of infection.
Methods
The bactericidal activities of LL-37, ceragenins CSA-13, CSA-90 and CSA-92 and the spermine-conjugated dexamethasone derivative D2S were evaluated using MIC and MBC measurements. Gingival fibroblast counting, interleukin-8 (IL-8) and lactate dehydrogenase (LDH) release from keratinocytes (HaCat) were used to determine effects on cell growth, pro-inflammatory response and toxicity.
Results
All tested cationic lipids showed stronger bactericidal activity than LL-37. Incubation of Staphylococcus aureus with half the MIC of LL-37 led to the appearance of bacteria resistant to its bactericidal effects, but identical incubations with CSA-13 or D2S did not produce resistant bacteria. Cathelicidin LL-37 significantly increased the total number of gingival fibroblasts, but ceragenins and D2S did not alter gingival fibroblast growth. Cationic lipids showed no toxicity to HaCat cells at concentrations resulting in bacterial killing.
Conclusions
These data suggest that cationic lipids such as ceragenins warrant further testing as potential novel antibacterial agents.
doi:10.1093/jac/dks434
PMCID: PMC3566669  PMID: 23134677
oral infection; microbiological assay; ceragenins; D2S
11.  Darunavir is predominantly unbound to protein in cerebrospinal fluid and concentrations exceed the wild-type HIV-1 median 90% inhibitory concentration 
Objectives
Higher CSF antiretroviral concentrations may be associated with better control of HIV replication and neurocognitive performance, but only the unbound fraction of antiretrovirals is available to inhibit HIV. Therefore, the objective of this study was to determine total and unbound darunavir concentrations in CSF and compare findings with plasma concentrations as well as the wild-type HIV-1 90% inhibitory concentration (IC90).
Methods
Subjects with HIV infection were selected based on the use of darunavir-containing regimens with a twice-daily dosing schedule and availability of stored CSF and matched plasma. Total darunavir was measured by HPLC for plasma or liquid chromatography–tandem mass spectroscopy (LC/MS/MS) for CSF. Plasma unbound darunavir was measured by ultrafiltration and LC/MS/MS. CSF protein binding was determined by competitive binding exchange with radiolabelled darunavir.
Results
Twenty-nine matched CSF–plasma pairs were analysed and darunavir was detected in all CSF specimens (median total concentration 55.8 ng/mL), with a CSF unbound fraction of 93.5%. Median fractional penetrance was 1.4% of median total and 9.4% of median unbound plasma concentrations. Unbound darunavir concentrations in CSF exceeded the median IC90 for wild-type HIV in all subjects by a median of 20.6-fold, despite the relatively low fractional penetrance. Total darunavir concentrations in CSF correlated with both total and unbound darunavir concentrations in plasma.
Conclusions
Darunavir should contribute to the control of HIV replication in the CNS as a component of effective combination antiretroviral regimens.
doi:10.1093/jac/dks441
PMCID: PMC3566670  PMID: 23143899
HIV; antiretroviral therapy; central nervous system; protein binding
12.  Minimal inhibitory and mutant prevention concentrations of azithromycin, clarithromycin and erythromycin for clinical isolates of Streptococcus pneumoniae 
Background
Previous work showed a higher prevalence of macrolide/azalide resistance in provinces of Canada where azithromycin was the major treatment for Streptococcus pneumoniae as compared with regions where clarithromycin was the dominant treatment. These data provided a way to test the mutant selection window hypothesis, which predicts that the serum drug concentration (AUC24) relative to the mutant prevention concentration (MPC) would be higher for clarithromycin than for azithromycin.
Methods
The MIC and MPC were determined for 191 penicillin/macrolide-susceptible clinical isolates of S. pneumoniae with azithromycin, clarithromycin and erythromycin using agar plate assays.
Results
The MIC50/90 (mg/L) and MPC50/90 (mg/L), respectively, were as follows: azithromycin 0.13/0.25 and 1/4; clarithromycin 0.031/0.063 and 0.13/0.5; erythromycin 0.063/0.13 and 0.25/2. We calculated from published pharmacokinetic values that the AUC24/MPC90 for azithromycin was 0.85; for clarithromycin it was 96, and for erythromycin base and estolate it was 4 and 10, respectively. Thus the AUC24/MPC90 was about 50 times higher for clarithromycin than for azithromycin.
Conclusions
The elevated prevalence of azithromycin resistance may derive in part from a low value of AUC24/MPC90 and/or time above MPC, since previous work indicates that the number of prescriptions per person was similar in the geographical regions examined.
doi:10.1093/jac/dks461
PMCID: PMC3566671  PMID: 23169894
pneumococcus; azalide; macrolide; MPC
13.  Decreasing population selection rates of resistance mutation K65R over time in HIV-1 patients receiving combination therapy including tenofovir 
Objectives
The use of tenofovir is highly associated with the emergence of mutation K65R, which confers broad resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs), especially when tenofovir is combined with other NRTIs also selecting for K65R. Although recent HIV-1 treatment guidelines discouraging these combinations resulted in reduced K65R selection with tenofovir, updated information on the impact of currently recommended regimens on the population selection rate of K65R is presently lacking.
Methods
In this study, we evaluated changes over time in the selection rate of resistance mutation K65R in a large population of 2736 HIV-1-infected patients failing combination antiretroviral treatment between 2002 and 2010.
Results
The K65R resistance mutation was detected in 144 patients, a prevalence of 5.3%. A large majority of observed K65R cases were explained by the use of tenofovir, reflecting its wide use in clinical practice. However, changing patterns over time in NRTIs accompanying tenofovir resulted in a persistent decreasing probability of K65R selection by tenofovir-based therapy. The currently recommended NRTI combination tenofovir/emtricitabine was associated with a low probability of K65R emergence. For any given dual NRTI combination including tenofovir, higher selection rates of K65R were consistently observed with a non-nucleoside reverse transcriptase inhibitor than with a protease inhibitor as the third agent.
Discussion
Our finding of a stable time trend of K65R despite elevated use of tenofovir illustrates increased potency of current HIV-1 therapy including tenofovir.
doi:10.1093/jac/dks380
PMCID: PMC3543118  PMID: 23027713
virology; trend; treatment; guidelines; epidemiology
14.  Sequential combined treatment with allopurinol and benznidazole in the chronic phase of Trypanosoma cruzi infection: a pilot study 
Objectives
Even though the use of combined drugs has been proved to be effective in other chronic infections, assessment of combined treatment of antiparasitic drugs in human Chagas' disease has not been performed. Herein, a pilot study was conducted to evaluate the tolerance and side effects of a sequential combined treatment of two antiparasitic drugs, allopurinol and benznidazole, in the chronic phase of Trypanosoma cruzi infection.
Patients and methods
Changes in total and T. cruzi-specific T and B cells were monitored during a median follow-up of 36 months. Allopurinol was administered for 3 months (600 mg/day) followed by 30 days of benznidazole (5 mg/kg/day) in 11 T. cruzi-infected subjects.
Results
The combined sequential treatment of allopurinol and benznidazole was well tolerated. The levels of T. cruzi-specific antibodies significantly decreased after sequential combined treatment, as determined by conventional serology and by a multiplex assay using recombinant proteins. The frequency of T. cruzi-specific interferon-γ-producing T cells significantly increased after allopurinol treatment and decreased to background levels following benznidazole administration in a substantial proportion of subjects evaluated. The levels of total naive (CD45RA + CCR7 + CD62L+) CD4 + and CD8 + T cells were restored after allopurinol administration and maintained after completion of the combined drug protocol, along with a decrease in T cell activation in total peripheral CD4 + and CD8 + T cells.
Conclusions
This pilot study shows that the combination of allopurinol and benznidazole induces significant modifications in T and B cell responses indicative of a reduction in parasite burden, and sustains the feasibility of administration of two antiparasitic drugs in the chronic phase of Chagas' disease.
doi:10.1093/jac/dks390
PMCID: PMC3543119  PMID: 23104493
Chagas' disease; trypanosomiasis; chemotherapy; T cells
15.  Partial protection against multiple RT-SHIV162P3 vaginal challenge of rhesus macaques by a silicone elastomer vaginal ring releasing the NNRTI MC1220 
Objectives
The non-nucleoside reverse transcriptase inhibitor MC1220 has potent in vitro activity against HIV type 1 (HIV-1). A liposome gel formulation of MC1220 has previously been reported to partially protect rhesus macaques against vaginal challenge with a simian HIV (SHIV). Here, we describe the pre-clinical development of an MC1220-releasing silicone elastomer vaginal ring (SEVR), including pharmacokinetic (PK) and efficacy studies in macaques.
Methods
In vitro release studies were conducted on SEVRs loaded with 400 mg of MC1220, using simulated vaginal fluid (SVF, n = 4) and 1 : 1 isopropanol/water (IPA/H2O, n = 4) as release media. For PK evaluation, SEVRs were inserted into adult female macaques (n = 6) for 30 days. Following a 1week washout period, fresh rings were placed in the same animals, which were then challenged vaginally with RT-SHIV162P3 once weekly for 4 weeks.
Results
SEVRs released 1.66 and 101 mg of MC1220 into SVF and IPA/H2O, respectively, over 30 days, the differential reflecting the low aqueous solubility of the drug. In macaque PK studies, MC1220 was consistently detected in vaginal fluid (peak 845 ng/mL) and plasma (peak 0.91 ng/mL). Kaplan–Meier analysis over 9weeks showed significantly lower infection rates for animals given MC1220-containing SEVRs than placebo rings (hazard ratio 0.20, P = 0.0037).
Conclusions
An MC1220-releasing SEVR partially protected macaques from vaginal challenge. Such ring devices are a practical method for providing sustained, coitally independent protection against vaginal exposure to HIV-1.
doi:10.1093/jac/dks415
PMCID: PMC3543122  PMID: 23109186
HIV-1 microbicide; silicone elastomer vaginal ring; rhesus macaque; pharmacokinetics; RT-SHIV challenge
16.  Response to raltegravir-based salvage therapy in HIV-infected patients with hepatitis C virus or hepatitis B virus coinfection 
Objectives
To define the impact of coinfection with hepatitis B virus (HBV) or hepatitis C virus (HCV) on viroimmunological response to raltegravir-based salvage regimens that also include new HIV inhibitors such as maraviroc, darunavir and etravirine.
Methods
We used data from a national observational study of patients starting raltegravir-based regimens to compare virological suppression and CD4 cell change from baseline in patients with and without concomitant HBV or HCV infection.
Results
Overall, 275 patients (107 coinfected and 168 non-coinfected) were evaluated. Coinfected patients were more commonly former intravenous drug users and had a longer history of HIV infection and higher baseline aminotransferase levels. Both HIV-RNA and CD4 response were similar in the two groups. Mean time to first HIV-RNA copy number <50 copies/mL was 4.1 months (95% CI 3.5–4.6) in non-coinfected patients and 3.9 months (95% CI 3.3–4.5) in coinfected patients (hazard ratio 1.039, 95% CI 0.761–1.418, P = 0.766, log-rank test). The risk of developing new grade 3–4 hepatic adverse events was significantly higher in coinfected patients (hazard ratio 1.779, 95% CI 1.123–2.817, P = 0.009). The two groups of coinfected and non-coinfected patients had similar rates of interruption of any baseline drug (hazard ratio 1.075, 95% CI 0.649–1.781, P = 0.776) and of raltegravir (hazard ratio 1.520, 95% CI 0.671–3.447, P = 0.311). Few AIDS-defining events and deaths occurred.
Conclusions
Viroimmunological response to regimens based on raltegravir and other recent anti-HIV inhibitors is not negatively affected by coinfection with HBV or HCV. Liver toxicity, either pre-existing or new, is more common in coinfected patients, but with no increased risk of treatment interruption.
doi:10.1093/jac/dks341
PMCID: PMC3522446  PMID: 22984206
antiretroviral therapy; HIV-1; HBV; HCV; integrase inhibitors; viral hepatitis; viral load; CD4 response; darunavir; maraviroc; etravirine; HIV resistance; liver disease
17.  Rapid single-colony whole-genome sequencing of bacterial pathogens 
Objectives
As a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to generate DNA libraries for whole-genome sequencing directly from single bacterial colonies grown on primary culture plates.
Methods
We developed our protocol using single colonies of 17 bacterial pathogens responsible for severe human infection that were grown using standard diagnostic media and incubation conditions. We then applied this method to four clinical scenarios that currently require time-consuming reference laboratory tests: full identification and genotyping of salmonellae; identification of blaNDM-1, a highly transmissible carbapenemase resistance gene, in Klebsiella pneumoniae; detection of genes encoding staphylococcal toxins associated with specific disease syndromes; and monitoring of vaccine targets to detect vaccine escape in Neisseria meningitidis.
Results
We validated our single-colony whole-genome sequencing protocol for all 40 combinations of pathogen and selective, non-selective or indicator media tested in this study. Moreover, we demonstrated the clinical value of this method compared with current reference laboratory tests.
Conclusions
This advance will facilitate the implementation of whole-genome sequencing into diagnostic and public health microbiology.
doi:10.1093/jac/dkt494
PMCID: PMC3977605  PMID: 24370932
infectious diseases; antibiotic resistance; typing
18.  Effectiveness of neuraminidase inhibitors in preventing hospitalization during the H1N1 influenza pandemic in British Columbia, Canada 
Objectives
In British Columbia (BC), Canada, neuraminidase inhibitors (NIs) were publicly funded during the 2009 A(H1N1)pdm09 pandemic for treatment of high-risk patients and/or anyone with moderate-to-severe illness. We assessed antiviral effectiveness (AVE) against hospitalization in that context.
Methods
A population-based cohort study was conducted using linked administrative data. The cohort included all individuals living in BC during the study period (1 September to 31 December 2009) with a diagnostic code consistent with influenza or pandemic H1N1. The main study period pertained to the second-wave A(H1N1)pdm09 circulation (1 October to 31 December 2009), with sensitivity analyses around the more specific pandemic peak (18 October to 7 November). Exposure was defined by same-day NI prescription. The main outcome was all-cause hospitalization within 14 days of the outpatient influenza diagnosis. Cox proportional hazards models assessed AVE with 1 : 1 propensity-score matching and covariate adjustment.
Results
After matching, there were 304/58 061 NI-exposed and 345/58 061 unexposed patients hospitalized during the main study period. The very young [<6 months (35.0; 95% CI 16.7–73.4)], the old [65–79 years (13.7; 95% CI 10.1–18.6)] and the very old [≥80 years (38.7; 95% CI 26.6–56.5)] had the highest hospitalization rate per 1000 patients overall. Fully adjusted AVE against all-cause hospitalization during the main study period was 16% (95% CI 2%–28%), similar to the pandemic peak (15%; 95% CI −4%–30%).
Conclusions
The use of NIs was associated with modest protection against hospitalization during the 2009 pandemic, but appeared underutilized in affected age groups with the highest hospitalization risk.
doi:10.1093/jac/dkt496
PMCID: PMC3977606  PMID: 24346762
oseltamivir; zanamivir; antivirals; mortality; population-based; cohort
19.  Characterization of HIV-1 antiretroviral drug resistance after second-line treatment failure in Mali, a limited-resources setting 
Journal of Antimicrobial Chemotherapy  2012;67(12):2943-2948.
Objectives
We describe the outcomes of second-line drug resistance profiles and predict the efficacy of drugs for third-line therapy in patients monitored without the benefit of plasma HIV-1 RNA viral load (VL) or resistance testing.
Methods
We recruited 106 HIV-1-infected patients after second-line treatment failure in Mali. VL was determined by the Abbott RealTime system and the resistance by the ViroSeq HIV-1 genotyping system. The resistance testing was interpreted using the latest version of the Stanford algorithm.
Results
Among the 106 patients, 93 had isolates successfully sequenced. The median age, VL and CD4 cells were respectively 35 years, 72 000 copies/mL and 146 cells/mm3. Patients were exposed to a median of 4 years of treatment and to six antiretrovirals. We found 20% of wild-type viruses. Resistance to etravirine was noted in 38%, to lopinavir in 25% and to darunavir in 12%. The duration of prior nucleos(t)ide reverse transcriptase inhibitor exposure was associated with resistance to abacavir (P < 0.0001) and tenofovir (P = 0.0001), and duration of prior protease inhibitor treatment with resistance to lopinavir (P < 0.0001) and darunavir (P = 0.06).
Conclusion
Long duration of therapy prior to failure was associated with high levels of resistance and is directly related to limited access to VL monitoring and delayed switches to second-line treatment, precluding efficacy of drugs for third-line therapy. This study underlines the need for governments and public health organizations to recommend the use of VL monitoring and also the availability of darunavir and raltegravir for third-line therapies in the context of limited-resource settings.
doi:10.1093/jac/dks310
PMCID: PMC3584968  PMID: 22888273
resistance; third-line; Africa
20.  Magic bullets for the 21st century: the reemergence of immunotherapy for multi- and pan-resistant microbes 
Journal of Antimicrobial Chemotherapy  2012;67(12):2785-2787.
In our current world, antibiotic resistance among pathogenic microbes keeps getting worse with few new antibiotics being pursued by pharmaceutical companies. Modern-day immunotherapies, reminiscent of the serotherapy approaches used in the early days of antimicrobial treatments, are a potential counter-measure, but are usually limited by the narrow spectrum against target antigens. Surprisingly, many multidrug-resistant (MDR) bacteria share a common surface polysaccharide, poly-β-1,6-N-acetylglucosamine (PNAG). Natural antibodies to PNAG are present in normal human sera, but are not protective. However, human monoclonal antibodies (MAbs) or polyclonal antisera raised to a deacetylated glycoform of PNAG mediate opsonic killing and protect mice against infections due to all PNAG-positive MDR pathogens tested. An MAb is currently in Phase II clinical trials. These discoveries could lead to utilization of antibodies to PNAG for either therapeutic use in patients infected by PNAG-producing MDR bacteria or prophylactic use in patients at risk of developing MDR infections.
doi:10.1093/jac/dks335
PMCID: PMC3695597  PMID: 22899807
poly-N-acetylglucosamine; PNAG; broad-spectrum vaccines; antibiotic resistance
21.  Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity 
Journal of Antimicrobial Chemotherapy  2012;67(12):2870-2872.
Background
Retrocyclins are cyclic antimicrobial peptides that have been shown to be both broadly active and safe in animal models. RC-101, a synthetic retrocyclin, targets important human pathogens and is a candidate vaginal microbicide. Its activity against microbes associated with bacterial vaginosis is unknown.
Methods
We investigated the effect of RC-101 on toxin activity, bacterial growth and biofilm formation of Gardnerella vaginalis in vitro.
Results
RC-101 potently inhibits the cytolytic activity of vaginolysin, the Gardnerella vaginalis toxin, on both erythrocytes and nucleated cells. RC-101 lacks inhibitory activity against planktonic G. vaginalis but markedly decreases biofilm formation.
Conclusions
These dual properties, toxin inhibition and biofilm retardation, justify further exploration of RC-101 as a candidate agent for bacterial vaginosis prevention.
doi:10.1093/jac/dks305
PMCID: PMC3494843  PMID: 22855857
defensin; vaginolysin; bacterial vaginosis; biofilm
22.  3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I 
Journal of Antimicrobial Chemotherapy  2012;67(12):2882-2891.
Objectives
Antibiotic resistance in bacterial pathogens is a serious clinical problem. Novel targets are needed to combat increasing drug resistance in Escherichia coli. Our objective is to demonstrate that 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2yl]-1H-benzimidazole (DMA) inhibits E. coli DNA topoisomerase I more strongly than human topoisomerase I. In addition, DMA is non-toxic to mammalian cells at antibiotic dosage level.
Methods
In the present study, we have established DMA as an antibacterial compound by determining MICs, post-antibiotic effects (PAEs) and MBCs for different standard as well as clinical strains of E. coli. We have described the differential catalytic inhibitory mechanism of bis-benzimidazole, DMA, for human and E. coli topoisomerase I and topoisomerase II by performing different assays, including relaxation assays, cleavage–religation assays, DNA unwinding assays, ethidium bromide displacement assays, decatenation assays and DNA gyrase supercoiling assays.
Results
DMA significantly inhibited bacterial growth at a very low concentration, but did not affect human cell viability at higher concentrations. Activity assays showed that it preferentially targeted E. coli topoisomerase I over human topoisomerase I, topoisomerase II and gyrase. Cleavage–religation assays confirmed DMA as a poison inhibitor of E. coli topoisomerase I. This study illuminates new properties of DMA, which may be further modified to develop an efficient topoisomerase inhibitor that is selective towards bacterial topoisomerase I.
Conclusions
This is the first report of a bis-benzimidazole acting as an E. coli topoisomerase I inhibitor. DMA is a safe, non-cytotoxic molecule to human cells at concentrations that are needed for antibacterial activity.
doi:10.1093/jac/dks322
PMCID: PMC3494844  PMID: 22945915
Hoechst 33342; DMA; MICs; MBCs; PAEs
23.  The newly described mecA homologue, mecALGA251, is present in methicillin-resistant Staphylococcus aureus isolates from a diverse range of host species 
Journal of Antimicrobial Chemotherapy  2012;67(12):2809-2813.
Objectives
A previously unidentified mecA homologue, mecALGA251, has recently been described in methicillin-resistant Staphylococcus aureus (MRSA) from humans and dairy cattle. The origin and epidemiology of this novel homologue are unclear. The objective of this study was to provide basic descriptive information of MRSA isolates harbouring mecALGA251 from a range of host animal species.
Methods
A number of S. aureus isolates from historical animal isolate collections were chosen for investigation based on their similarity to known mecALGA251 MRSA isolates. The presence of mecALGA251 was determined using a multiplex PCR and antimicrobial susceptibility testing performed by disc diffusion.
Results
MRSA harbouring mecALGA251 were found in isolates from a domestic dog, brown rats, a rabbit, a common seal, sheep and a chaffinch. All of the isolates were phenotypically MRSA, although this depended on which test was used; some isolates would be considered susceptible with certain assays. All isolates were susceptible to linezolid, rifampicin, kanamycin, norfloxacin, erythromycin, clindamycin, fusidic acid, tetracycline, trimethoprim/sulfamethoxazole and mupirocin. Five multilocus sequence types were represented (2273, 130, 425, 1764 and 1245) and six spa types (t208, t6293, t742, t6594, t7914 and t843).
Conclusions
The discovery of MRSA isolates possessing mecALGA251 from a diverse range of host species, including different taxonomic classes, has important implications for the diagnosis of MRSA in these species and our understanding of the epidemiology of this novel mecA homologue.
doi:10.1093/jac/dks329
PMCID: PMC3494845  PMID: 22941897
animal infections; animal reservoirs; wildlife; MRSA
24.  Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs 
Objectives
Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs.
Methods
The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants.
Results
All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical.
Conclusions
TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
doi:10.1093/jac/dkt442
PMCID: PMC3922157  PMID: 24235095
drug transport; protozoan; parasite; resistance mutation; aquaporin
25.  Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene 
Objectives
Methicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to β-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to β-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant β-lactam antibiotics.
Methods
The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays.
Results
We identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types.
Conclusions
This work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for β-lactam resistance in MRSA that may need to be considered by diagnostic laboratories.
doi:10.1093/jac/dkt418
PMCID: PMC3922151  PMID: 24216768
β-lactams; MRSA; mecA; mecC

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