Genotypic and phenotypic tests were used to evaluate the resistance of virus isolated from cell culture in the presence of the CCR5 inhibitor TAK-779.

Mutations conferring resistance appeared in the gp120 sequence but were not confined to the V3 loop region, and both strains had a different mutation pattern. Recombination of the env gene of the BaL-derived resistant virus into the HIV-1 HXB2 wild-type backbone conferred resistance to TAK-779 and cross-resistance to maraviroc, with 63- and 11-fold changes in their EC₅₀ (50% effective concentration), respectively, together with an apparent reduction of the maximal plateau inhibition (MPI) of TAK-779 but not of maraviroc. Conversely, the resistant CI viruses showed an ~50% reduction in MPI for both TAK-779 and maraviroc.

We confirm that different pathways to the generation of CCR5 drug resistance/cross-resistance may occur that strongly depend on cell culture conditions, CCR5 availability and the genetic background of the HIV strain. Our study provides complementary information to understand the complexity of resistance to CCR5 antagonists.

Clonal genotypic analyses were performed on sequential HIV-1 integrase sequences amplified from 11 failing patients and sampled every 4–24 weeks for up to 64 weeks. Fully replicating recombinant viruses were generated using modified vectors in which selected viral integrase genes amplified from patients’ plasma were cloned. rRC was measured by a novel multiple cycle competition assay. Resistance to raltegravir and the rRC of resistant HIV-1 variants selected in vivo were evaluated in purified CD4+ T cells.

In all of the patients but one, failure was associated with selection of mutations in positions 143, 148 or 155. Unlike mutations at position 143 (Y143S/K/R), identified alone or in combination with others, mutations at position 148 and 155 were always found in combination. A wide range of resistance levels to raltegravir [from 10- to 770-fold change in 50% inhibitory concentration (IC₅₀) compared with baseline] was observed using recombinant viral clones. Finally, rRC was not significantly altered in highly resistant variants.

Two patterns of viral evolution were observed in the resistant viral populations, driving the variants towards a fast (most of them with G140S + Q148H mutations) or progressive increase in resistance to raltegravir. These results may have implications either for the evaluation of genotypic results, or for the correct clinical use of the compound.

Twenty-five patients starting a raltegravir-based regimen were included. Total HIV-1 DNA and 2-LTR DNA were quantified at baseline and in follow-up samples up to month 12. The effect of raltegravir on the formation of 2-LTR circles was evaluated in HeLa P4 cells. The effect of raltegravir was also investigated by sequence analysis of the 2-LTR circle junctions.

Among 21 patients with undetectable 2-LTR DNA at baseline, 7 had detectable 2-LTR DNA during the follow-up. Three of four patients with detectable 2-LTR DNA at baseline had undetectable 2-LTR DNA during the follow-up (P = 0.27). The mean 2-LTR level increased significantly (+0.07 log₁₀/month, P = 0.02) in raltegravir-treated patients, and a 2-LTR increase was also observed in raltegravir-treated HeLa P4 cells, with a peak at 3 days post-infection. 2-LTR DNA showed a high prevalence of deletions ex vivo (64.5%) and in vitro (50%) in the presence of raltegravir, which was not statistically different from the prevalence in untreated patients or cells.

In antiretroviral-experienced patients receiving raltegravir, 2-LTR DNA increased while total HIV-1 DNA decreased over time. The frequent rearrangements found in 2-LTR sequences warrant further investigations to determine the dynamics of evolution of unintegrated HIV-1 DNA.

PCR–restriction fragment length polymorphism of polymorphic codons of the dhfr gene (51, 59 and 108) and the dhps gene (436, 437 and 540) was performed in matched peripheral and placental blood samples.

The proportion of multiple mutations was high (98%), and was not different between women with and without a history of intermittent preventive treatment with sulfadoxine/pyrimethamine (IPTp/SP). The prevalence of triple dhfr mutation was 80%, and that of quadruple and quintuple mutations was 53% and 22%, respectively. The Glu540 mutation was present in two isolates. The concordance of resistant alleles in matched peripheral and placental isolates was >90% for both genes.

These findings underline the need for a regular assessment of the relationship between the presence of resistant isolates and in vitro/in vivo IPTp/SP efficacy, and evaluation of an alternative drug.

Two paediatric males with cystic fibrosis had sputum samples quantitatively cultured during hospitalization. After the isolation of MRSA from both patients, oral treatment with 300 mg linezolid twice daily was initiated for periods of 1–2 months separated by up to 6 months. Isolates cultured 9 months after the start of treatment were tested for resistance to linezolid by agar dilution (BSAC). Resistant isolates were examined for 23S rDNA mutations, and typed by phage and macrorestriction with SmaI. Isolates from follow-up sputum samples were obtained until 44–51 months after treatment with linezolid.

Colonization with MRSA was at a density of ~10⁶ cfu/mL sputum for both subjects. Initial isolates were susceptible to linezolid, but, 9 months later, isolates from both patients were resistant (MICs > 16 mg/L). Both isolates were epidemic MRSA-16 variant A1 (ST36-MRSA-II), which is widespread in UK hospitals. Both isolates were heterozygous for a G2576T mutation in their 23S rDNA genes, but one was resistant to fusidic acid and tetracycline. In follow-up sampling, the younger patient yielded linezolid-resistant EMRSA-16 for a further 42 months, whilst the other lost the linezolid-resistant MRSA and had alternately Pseudomonas aeruginosa or linezolid-susceptible EMRSA-16 variant A1 isolated over 35 further months.

Linezolid resistance emerged in two isolates of ST36 MRSA colonizing the lungs of two paediatric cystic fibrosis patients. Subtherapeutic levels of linezolid may have facilitated the selection of resistance.

Consecutive MRSA isolates collected in 2001, 2003, 2005 and 2007 by the BSAC Bacteraemia Surveillance Programme were categorized to multilocus sequence typing (MLST) clonal complex and to SCCmec type by PCR. MICs were determined by the BSAC method. Data trends were tested for significance using a generalized linear regression model.

Collectively, EMRSA-15 and EMRSA-16 consistently accounted for ~95% of MRSA studied between 2001 and 2007, but the proportions of EMRSA-16 declined from 21.4% in 2001 to 9% in 2007 (P < 0.05), whilst the proportion of EMRSA-15 rose commensurately, accounting for 85% of MRSA in 2007. Ciprofloxacin and erythromycin resistance were common amongst both EMRSA-15 and EMRSA-16.

EMRSA-15 and EMRSA-16 remain the main MRSA strains in bacteraemia in the UK, but the proportion of EMRSA-16 declined from the late 1990s, thus preceding the general decline in MRSA bacteraemias that began in the middle of the present decade.

During the study period (September 2004–February 2007), isolates of S. pneumoniae cultured by the LTHTR microbiology laboratory were examined by Etest to determine MICs of levofloxacin. Isolates from patients in whom there was a shift towards colonization with S. pneumoniae of reduced levofloxacin susceptibility were further characterized by serotyping, multilocus sequence typing (MLST) and sequencing of parC and gyrA genes.

Eight hundred and sixty-five isolates were collected; however, 772 isolates from 652 patients were recoverable; 412 (53.4%) came from hospitalized patients, 12 (1.6%) were resistant to levofloxacin according to the BSAC breakpoint (>2 mg/L) and 29 (3.8%) had MICs at the breakpoint (MIC = 2 mg/L). Of six patients in whom there was a shift towards isolates with reduced levofloxacin susceptibility, five had acquired new distinct strains. One patient, who had a parC mutation (Ser79Phe) in the original susceptible isolate and an additional second-step mutation in the gyrA gene (Ser81Phe) of the later resistant one, had isolates belonging to the same pneumococcal clone.

S. pneumoniae resistance to levofloxacin was uncommon and we managed to identify only one case of probable in vivo resistance development in the 2.5 years of the study. Strain replacement accounted for the majority of incidences where there was an apparent shift towards colonization with isolates of reduced levofloxacin susceptibility.

In total, 579 clinical Escherichia coli isolates were subjected to antimicrobial susceptibility testing, screening for qnr alleles, qepA and aac-(6')-Ib-cr by PCR amplification and DNA sequence analysis. Isolates containing transferable quinolone resistance determinants were further characterized by mutation analysis in the quinolone resistance determining regions (QRDRs) of GyrA and ParC, phylogenetic typing and PFGE to determine their genetic relatedness.

After PCR screening and sequence analysis, transferable quinolone resistance determinants were identified in 74 of 579 E. coli isolates (12.8%). The antimicrobial resistance profiles and phylogenetic groups differed between isolates containing different categories of transferable quinolone resistance determinant. Most of the isolates containing qepA alone were highly resistant to ciprofloxacin (MIC ≥ 512 mg/L) and belonged to phylogenetic group D (22/25), while most of the isolates containing aac-(6')-Ib-cr alone belonged to phylogenetic group A (17/35) or D (16/35). Of 74 E. coli isolates containing transferable quinolone resistance determinants, 69 PFGE patterns and 19 clusters were identified.

The great genetic variation of E. coli hosts containing transferable quinolone resistance determinants demonstrated the high transmission capacity of these mechanisms. It is urgent to characterize and block their transmission routes in order that the utility of quinolones is preserved.

Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporins and resistance to cefoxitin were collected from the Department of Clinical Microbiology at Hvidovre Hospital, Denmark, in 2006. The AmpC disc test was used to confirm expression of AmpC, and test-positive strains were selected for further antimicrobial susceptibility testing and molecular characterization. Hyperproduction of AmpC β-lactamase was confirmed by isoelectric focusing (IEF). The presence of a plasmid-mediated ampC gene (pAmpC) was detected by multiplex PCR. The promoter and the entire reading frame of the chromosomal ampC gene were sequenced to identify promoter mutations associated with hyperproduction and gene mutations associated with extended-spectrum AmpC (ESAC) β-lactamase activity.

Twenty-four isolates exhibited a positive AmpC disc test. IEF confirmed AmpC expression in all isolates except one. Four isolates contained a bla_CMY-2 gene. These were not clonally related by multilocus sequence typing (MLST). The remaining isolates all had mutations or insertions in the promoter region, which could explain increased expression of the chromosomal AmpC enzyme. Mutations in the ampC gene associated with extended activity were rare and did not cause resistance to cefepime. Sequencing of ampC showed that most isolates were not clonally related.

E. coli expressing an AmpC phenotype occur sporadically and cause significant resistance to cephalosporins. The majority of these are hyperproducing chromosomal ampC although some isolates have acquired pAmpC.

A survey was carried out to investigate the molecular epidemiology and genetic characteristics of clinical ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates originating from the Dr. Soetomo Academic Hospital in Surabaya, Indonesia, over a 4 month period (January to April 2005). ESBLs were characterized by isoelectric focusing and PCR assays. Clonality of the isolates was assessed by PFGE and repetitive-sequence-based PCR (rep-PCR). Phylogenetic grouping was performed among CTX-M-15-producing E. coli.

In total, 73 consecutive non-duplicate ESBL-positive E. coli and 72 K. pneumoniae strains were isolated. The bla_CTX-M-15 gene was found to be highly prevalent (69/73 strains, 94.5%) among the 73 ESBL-positive E. coli isolates. The gene was detected in both clonal and non-clonal isolates, as defined by PFGE and rep-PCR. Sixteen CTX-M-15-positive E. coli could be assigned to a single rep-PCR type and phylogenetic group B2 and belonged to the well-known O25b-ST131 clone. Among the 72 ESBL-positive K. pneumoniae isolates, bla_CTX-M-15 was again the most prevalent ESBL (40/72, 55.6%). Several SHV-type enzymes were also frequently detected: SHV-5 (n = 28); SHV-12 (n = 13); and SHV-2 (n = 6). TEM-type ESBLs were not detected in any of the isolates.

Indonesia is another developing country affected by the emergence and spread of bacterial strains harbouring ESBL genes, including the CTX-M-15-producing B2-E. coli O25b-ST131 clone.

A PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the conserved 535 bp region of the H. pylori 16S rRNA genes rrnA/B (between nucleotides 710 and 1245) using HinfI was followed by DNA sequencing of the same fragment obtained from tetracycline-resistant H. pylori clinical isolates.

The PCR-RFLP assay revealed that the tetracycline-resistant H. pylori isolates lacked the AGA926–928->TTC substitution. In contrast, DNA sequencing of the 535 bp PCR fragment from 11 tetracycline-resistant H. pylori Chilean clinical isolates showed an association of low-level tetracycline resistance with 1 bp (A928C) or 2 bp (AG926–927->GT and/or A926G/A928C) substitutions in both 16S rRNA genes.

The PCR-RFLP (HinfI) assay alone is unreliable for the detection of tetracycline resistance in Chilean clinical isolates of H. pylori. To that end, it must be complemented by sequencing of the 535 bp PCR fragment.

Etests were used for susceptibility testing and screening for MBLs, confirmed through bla_VIM PCRs and sequencing. Clonal relatedness was evaluated by PFGE and multilocus sequence typing (MLST). MBL-carrying plasmids were characterized by restriction fragment length polymorphism, Southern blot and electroporation. MBL genetic elements were studied by cloning and sequencing.

MBL-producing P. putida was detected in eight patients (one clone each; two harbouring bla_VIM-1 and six harbouring bla_VIM-2), representing 14% of all the infections by the P. putida/fluorescens group. MBLs were detected in only 0.3% of P. aeruginosa infections (11 patients) during the same period. PFGE revealed four P. aeruginosa clones: one producing bla_VIM-13 (two patients); and three producing bla_VIM-2 (two patients, six patients and one patient, respectively). MLST indicated that the VIM-13 clone was the internationally spread sequence type (ST)235, while the major VIM-2 lineage corresponded to ST179, which is associated with chronic respiratory infections. The VIM-1 integron was shown to have both plasmid and chromosomal location, while the VIM-13 integron was only chromosomal. The VIM-2 integron was located in the same transposon (Tn402/Tn5053-like) in all P. aeruginosa and P. putida isolates, suggesting its crucial role in the dissemination of VIM-2.

The high diversity and proportion of MBL-positive P. putida suggests an environmental reservoir of these resistance determinants. Dissemination of these multidrug resistance elements to successful P. aeruginosa clones presents a major epidemiological and clinical threat.

Expression of RND-type efflux pump genes was analysed by real-time RT–PCR. Laboratory mutants were selected by exposure to either tigecycline or tetracycline in vitro. Efflux pump genes were inactivated by suicide plasmids containing the R6K origin of replication.

Higher tigecycline MICs correlated with elevated expression of the RND-type efflux pump genes sdeXY. Inactivation of sdeY or the outer membrane component gene hasF reduced MICs of tigecycline, tetracycline, ciprofloxacin and cefpirome to below those for strain NCTC 10211. A tetracycline-selected laboratory mutant also showed increases in sdeXY expression and tigecycline MIC.

Up-regulation of endogenous SdeXY–HasF-mediated efflux is associated with tigecycline resistance in S. marcescens along with MIC rises for tetracycline, ciprofloxacin and cefpirome. Inactivation of this efflux system reduced MICs of those compounds to below those for strain NCTC 10211.

PCR for Smqnr alleles was carried out on a selection of S. maltophilia from clinical specimens, and amplicons were cloned and transformed in E. coli TOP10 cells. Transformed colonies carrying the plasmid were tested for susceptibility to a range of quinolones by MIC determination. DNA sequences were determined and translated peptide sequences compared with known SmQnr sequences.

Thirteen isolates were found to contain Smqnr alleles, of which six corresponded to previously identified Smqnr sequences, while seven were novel variants. Increases in quinolone MICs compared with wild-type E. coli TOP10 were seen for all strains transformed with Smqnr alleles.

There is considerable diversity within Smqnr alleles. S. maltophilia may be a significant reservoir for the dissemination of quinolone resistance elements to Enterobacteriaceae.

Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify β-lactamase genes.

Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC β-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC β-lactamase (DHA-1).

We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered β-lactamases. This method allowed direct sequencing from the PCR products.

Susceptibility to trivalent antimony as determined in vitro with intracellular amastigotes from both visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients correlated well with the clinical response. Higher accumulation of trivalent antimony (SbIII) was observed in all susceptible isolates compared with resistant isolates. Reduced accumulation of SbIII correlated, with a few exceptions, with down-regulation of AQP1 RNA as determined by real-time PCR. Cloning and sequencing of the AQP1 gene from both VL and PKDL isolates showed sequence variation in four of the clinical isolates. None of the isolates had an alteration of Glu152 and Arg230, which have been previously shown to affect metalloid transport. Transfection of the AQP1 gene in a sodium antimony gluconate-resistant field isolate conferred susceptibility to the resistant isolate.

Our studies indicate genetic variation in VL and PKDL isolates. Down-regulation of AQP1 correlates well with clinical drug resistance in a majority of Indian VL and PKDL isolates. AQP1 gene expression at both the genetic and transcriptional level showed positive correlation with SbIII accumulation, with some exceptions.

Mouse peritoneal macrophages (PEMs), mouse bone marrow-derived macrophages (BMM), human peripheral blood monocyte-derived macrophages (PBM) and differentiated THP-1 cells were infected with L. donovani. Cultures were incubated with sodium stibogluconate, amphotericin B deoxycholate (Fungizone^®), miltefosine or paromomycin sulphate over six concentrations in 3-fold serial dilutions for 5 days. Analysis was based on percentage inhibition of infected macrophages and EC₅₀/EC₉₀ values estimated using sigmoidal curve-fitting.

The rank order of drug activity was the same in the different macrophage populations: amphotericin B > miltefosine > sodium stibogluconate > paromomycin. However, significant (P < 0.05) differences were observed between populations. Amphotericin B was more active in PEMs and BMM (EC₅₀ 0.02–0.06 µM) compared with PBM and differentiated THP-1 cells (EC₅₀ 0.08–0.40 µM) and miltefosine was more active in PBM (EC₅₀ 0.16–0.74 µM) compared with PEMs and BMM (EC₅₀ 2.60–7.67 µM). Sodium stibogluconate displayed highest activity in PBM (EC₅₀ 1.38–1.89 µg Sb^v/mL), followed by PEMs (EC₅₀ 21.75–27.79 µg Sb^v/mL) and BMM and differentiated THP-1 cells (EC₅₀ 28.96–112.77 µg Sb^v/mL). Paromomycin showed highest activity in PBM (EC₅₀ 80.03–104.38 µM) and PEMs (EC₅₀ 75.42–201.63 µM).

In vitro activity of anti-leishmanial drugs is host cell dependent. This has implications for: (i) the evaluation of in vitro drug activity; (ii) the evaluation of drug susceptibility of clinical isolates; and (iii) the standardization of anti-leishmanial drug assays.

Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy.

We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure–activity relationship analysis on the thiazolide compounds used in this study.

The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

E. coli was treated with thiourea plus 2,2'-bipyridyl to block hydroxyl radical accumulation, and the effect on quinolone lethality was measured for quinolones that distinguished the two lethal pathways: oxolinic acid requires protein synthesis to kill E. coli, while PD161144, a C-8-methoxy fluoroquinolone, does not. The lethal activity of another fluoroquinolone, moxifloxacin, was partially blocked by the presence of chloramphenicol, an inhibitor of protein synthesis. That feature made it possible to determine whether the effects of chloramphenicol and thiourea plus 2,2'-bipyridyl were additive.

Lethal activity of oxolinic acid was completely blocked by thiourea plus 2,2'-bipyridyl and by chloramphenicol. In contrast, PD161144 lethality was unaffected by these treatments. With moxifloxacin, both chloramphenicol and thiourea plus 2,2'-bipyridyl separately exhibited the same partial inhibition of quinolone lethality. No additivity in protection from moxifloxacin lethality was observed when thiourea, 2,2'-bipyridyl and chloramphenicol were combined and compared with the effect of chloramphenicol or thiourea plus 2,2'-bipyridyl used separately.

Inhibitor studies indicated that hydroxyl radical action contributes to quinolone-mediated cell death occurring via the chloramphenicol-sensitive lethal pathway but not via the chloramphenicol-insensitive pathway.

Two methicillin-susceptible (MSSA) ATCC strains and two methicillin-resistant (MRSA) clinical strains were used. New Zealand white rabbits were surgically implanted with a silicone intravenous catheter. Infection was induced by filling and locking the catheter with 0.3 mL of broth culture containing S. aureus, with turbidity equivalent to that of a 0.5 McFarland standard. Eighteen hours later the antibiotic-lock technique was started and continued for 24 h. Treatment groups were: control without treatment; 2000 mg/L linezolid; 2000 mg/L vancomycin; 2000 mg/L ciprofloxacin; and 40 000 mg/L gentamicin.

Linezolid and vancomycin showed equivalent activity, achieving significant reductions in log₁₀ cfu recovered from catheter tips in one MSSA strain (>1.12) and one MRSA strain (>0.77) as compared with controls (P < 0.05). Ciprofloxacin achieved significant log₁₀ cfu reductions in MSSA strains relative to controls (>2.51, P < 0.01). In one MSSA strain, ciprofloxacin showed a larger reduction in log₁₀ cfu than linezolid or vancomycin (P < 0.01). Gentamicin was the only antibiotic achieving negative catheter tip cultures (up to 87.5% in MSSA and up to 40% in MRSA, P < 0.01), and showed the greatest log₁₀ cfu reduction compared with controls (>4.25 in MSSA and >2.93 in MRSA, P < 0.05) and significant differences relative to the remaining treatment groups (P < 0.05 in both MSSA and MRSA).

Gentamicin showed the highest activity against both MSSA and MRSA biofilms.

We evaluated the effect of formononetin on trophozoite attachment to glass, to intestinal epithelial cell layers in vitro and to murine small intestinal explants, and on the intestinal load in mice.

We found that the isoflavone formononetin inhibits both attachment and flagellar motility within minutes and reduces the trophozoite load of Giardia in mice within 1.5 h after treatment.

The antigiardial activity of formononetin is at least partially due to its capacity to rapidly detach trophozoites.

Livers and spleens previously obtained from VL-infected BALB/c mice (following intravenous AmBisome^® or oral AmB treatments) were analysed for AmB concentrations. Then, non-infected BALB/c mice were divided into three treatment groups: a single dose of intravenous AmBisome^® (2 mg/kg, n = 5); and oral AmB every 12 h for 5 days (10 mg/kg, n = 6 and 20 mg/kg, n = 6). The animals were sacrificed 7 days after the initiation of the treatment and the livers and spleens were harvested for drug analysis by HPLC.

The single intravenous injection of AmBisome^® resulted in a 77-fold lower concentration of AmB in infected compared with non-infected liver tissue, while the difference in AmB concentration in the spleen was only 5-fold. The multiple dose oral administration of AmB resulted in a 3-fold lower concentration of AmB in infected compared with non-infected livers for both oral doses, while the differences in AmB concentrations in the spleen were not statistically different for the oral treatment groups.

VL significantly lowered the concentration of AmB in the liver and the spleen when compared with uninfected animals. This effect seems to correlate with the degree of infection of the tissue. In the case of the intravenous liposomal formulation (AmBisome^®), the differences between the infected and non-infected tissues are of a higher magnitude than in the case of orally administered AmB (iCo-009).

This Phase I, comparative, open-label, three-period, single-dose, crossover study was designed as a pilot study to exclude large (>40%) differences in the exposure to lopinavir. Single doses of medication, normalized to 400 mg of lopinavir, were administered on an empty stomach, 1 week apart. A 32 h pharmacokinetic curve was recorded. In an additional part of the study, in five of the same volunteers, a pharmacokinetic curve was recorded after administration of the Lopimune granules and Kaletra oral solution, both with food.

Twelve healthy subjects were enrolled (four females). The median (range) age, height and body weight were 24 (21–55) years, 1.79 (1.63–1.95) m and 72 (51–87) kg, respectively. The median [interquartile range (IQR)] AUC_0–t of lopinavir was 71.8 (48.8–93.5), 38.7 (28.7–52.2) and 58.7 (42.5–79.4) mg·h/L with Kaletra tablets, Lopimune granules and Lopimune paediatric tablets, all taken on an empty stomach, respectively. The respective C_max values were 7.2 (5.8–8.3), 4.6 (4.1–5.2) and 6.5 (5.0–7.1) mg/L after intake of the different formulations. When comparing the Lopimune formulations with the reference product Kaletra, for all parameters the differences were statistically significant (P ≤ 0.015). Ritonavir exposure was also lower after intake of the generic formulations versus Kaletra. When the five subjects took the Lopimune granules or Kaletra solution with food, the median (IQR) AUC_0–t of lopinavir was 58.5 (55.4–77.6) and 49.6 (39.1–58.1) mg·h/L, respectively.

Large differences in pharmacokinetic parameters can be excluded for Lopimune paediatric tablets when compared with the branded product and taken on an empty stomach, and also for Lopimune granules when these are taken with food.

Prospective, observational study of all antiretroviral-experienced HIV-infected patients who initiated raltegravir from January 2006 to January 2009 at a reference HIV clinic. Clinical data, laboratory parameters and liver stiffness measured at baseline, week 4 and every 3 months thereafter were collected. Chronic hepatitis C was defined as positive serum HCV-RNA. Grade 1–4 hepatotoxicity was defined following the AIDS Clinical Trials Group definition for liver enzyme elevations (LEEs). A control group of patients who initiated protease inhibitors (PIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) was examined similarly.

Data from 218 HIV-infected patients on raltegravir were analysed, 126 HIV mono-infected and 92 HIV/HCV co-infected patients. Any degree of LEEs occurred in 10 (7.9%) HIV mono-infected and 23 (25%) co-infected patients (relative risk 3.1; 95% confidence interval 2.9–3.4; P = 0.002). Severe hepatotoxicity (grade 3–4), however, was only seen in 3 (1.4%) patients, all co-infected with HCV. It occurred at months 1, 15 and 15, respectively. In all three subjects other reasons than raltegravir exposure most likely explained LEEs. Multivariate analysis revealed HCV co-infection as the only independent variable associated with any degree of hepatotoxicity on raltegravir (P = 0.03). Finally, the rate of LEEs in patients on raltegravir was lower than in those who were treated with PIs or NNRTIs.

LEEs are less frequent in patients treated with raltegravir than with other antiretroviral drug classes. However, HIV/HCV co-infected patients treated with raltegravir experienced LEEs more frequently than HIV mono-infected persons. In this series, LEEs in patients treated with raltegravir were uniformly mild and no cases of grade 3–4 hepatotoxicity could be directly attributed to the drug. These results reinforce the overall hepatic safety profile of raltegravir.

All HIV-positive patients living in Europe and Argentina recruited in EuroSIDA (1994–2006) were tested for serum HBV surface antigen (HBsAg). Chronic carriers were further characterized virologically at one central laboratory. Variables influencing HBV genotype distribution and viraemia were assessed using logistic regression.

From 16 505 HIV patients enrolled in EuroSIDA, 1179 (7.1%) were HBsAg positive, of whom 474 had specimens that allowed inclusion in the virological substudy. Overall 293 (62%) were treated with anti-HBV active antiretroviral drugs at the time of testing. Hepatitis delta virus superinfection was recognized in 14% and hepatitis C virus (HCV) antibodies in 27%. Serum HBV DNA was detectable in 315 (66.5%) and HBV genotyping gave results in 170 (35.9%) patients. HBV genotype distribution was as follows: A (72.9%), D (17.1%), G (1.8%), E (1.2%), F (1.2%) and C (0.6%); another 5.9% were co-infected with multiple HBV genotypes. In the multivariate analysis, the best predictor of HBV genotype A infection was risk exposure other than intravenous drug use, whereas predictors for detectable HBV viraemia were lower CD4 counts and lack of HCV antibodies.

A substantial proportion of HIV-positive patients with chronic hepatitis B show detectable HBV viraemia despite being treated with anti-HBV active antiretroviral drugs (mainly lamivudine). Low CD4 counts were associated with an independent higher risk of detectable HBV viraemia, which supports an earlier introduction of antiretroviral therapy, including anti-HBV drug(s) more potent than lamivudine.

HIV-infected patients with plasma viral load <400 copies/mL, fasted triglycerides from 2.3 to 11.4 mmol/L and/or fasted low-density lipoprotein (LDL)-cholesterol >4.1 mmol/L were randomized to switch the nucleoside reverse transcriptase inhibitor (NRTI) backbone to fixed-dose combination tenofovir disoproxil fumarate + emtricitabine or to maintain the baseline antiretroviral regimen (the control group). The study has been registered with ClinicalTrials.gov under the identifier NCT00323492.

Ninety-one patients were included in the intent-to-treat (ITT) analysis with triglycerides 2.4 mmol/L and LDL-cholesterol 4.0 mmol/L (median values). At week 12, the median changes from baseline of triglycerides were –0.5 mmol/L (–25%; n = 46) and –0.1 mmol/L (–6%; n = 45) in the tenofovir disoproxil fumarate + emtricitabine and control groups, respectively, indicating a difference of –0.4 mmol/L (P = 0.034) [95% confidence interval (CI): –0.9 to –0.0]. Similarly for LDL-cholesterol, changes of –0.4 mmol/L (–9%) and –0.1 mmol/L (–1%) were observed in the tenofovir disoproxil fumarate + emtricitabine and control groups, respectively, indicating a difference of –0.4 mmol/L (P = 0.031) [95% CI: –0.7 to –0.0]. The proportion of patients with LDL-cholesterol >4.1 mmol/L decreased from 48% at baseline to 26% at week 12 in the tenofovir disoproxil fumarate + emtricitabine group versus no change in the control group. No virological failure was observed during the study.

Switching to tenofovir disoproxil fumarate + emtricitabine in dyslipidaemic HIV-infected patients improves triglycerides and LDL-cholesterol.

Candidaemia episodes prospectively collected through a blood culture surveillance programme in a single institution. The study was divided into two periods of time, 1994–2003 (A) and 2004–2008 (B), according to the introduction of echinocandin treatment. Non-conditional logistic regression methods with mortality as the dependent variable were used.

Four hundred and thirty-three (3%) candidaemias out of 15 628 bloodstream infection episodes were analysed. Candida albicans was the most frequent species (211; 49%). Mortality was noted in 132 cases (30%). A total of 262 and 171 candidaemias were reported in period A and B, respectively. There were 94 deaths in period A (36%) and 38 in period B (22%, P = 0.03). Treatment in period A was amphotericin B in 89 patients (41 dead, 46%) and fluconazole in 151 (41 dead, 27%, P = 0.003). In period B, 113 patients received a triazole (26 dead, 23%), 30 an echinocandin (3 dead, 10%, P = 0.08) and 9 (0 dead) were treated with combined therapy (echinocandin and triazole). Mortality was higher in period A (94 dead, 36%) than in period B (38 dead, 27%), P = 0.03. Independent risk factors associated with mortality in period B were: age, chronic renal failure, ultimately or rapidly fatal prognosis of underlying disease and shock. Echinocandin alone or in combination therapy was associated with better outcome (odds ratio = 0.22, 95% confidence interval = 0.06–0.81, P = 0.02).

In patients with candidaemia, echinocandin therapy results in a better outcome.

We retrospectively collected data from all the cases of PJI that were managed with two-stage revision over a 4 year period. Patients were managed with an antibiotic-free period before reimplantation, in order to confirm, clinically and microbiologically, that infection was successfully treated.

One hundred and fifty-two cases were identified. The overall success rate (i.e. retention of the prosthesis over 5.75 years of follow-up) was 83%, but was 89% for first revisions and 73% for re-revisions [hazard ratio = 2.9, 95% confidence interval (CI) 1.2–7.4, P = 0.023]. Reimplantation microbiology was frequently positive (14%), but did not predict outcome (hazard ratio = 1.3, 95% CI 0.4–3.7, P = 0.6). Furthermore, most unplanned debridements following the first stage were carried out before antibiotics were stopped (25 versus 2 debridements).

We did not identify evidence supporting the use of an antibiotic-free period before reimplantation and routine reimplantation microbiology. Re-revision was associated with a significantly worse outcome.

A structured questionnaire survey was performed on the composition, organization and service activities of the AMT in all acute care and larger chronic care hospitals in the country in 2007. Descriptive statistics were stratified by duration of AMT funding.

Completed questionnaires were provided by 112 of 116 hospitals (response rate, 96.6%). Mutidisciplinary AMTs varied in size (mean 10, range 2–28 members). Antibiotic stewardship tools used by AMTs included: hospital antibiotic formulary (96.3% of hospitals); practice guidelines for antibiotic therapy and surgical prophylaxis (91.6% and 96.3%, respectively); list of ‘restricted’ antimicrobial agents (75.9%); concurrent review of antibiotic therapies (64.2%); de-escalation of therapy after a few days (63.9%); sequential intravenous/oral therapy for antibiotics with equivalent bioavailability (78.7%); dedicated antimicrobial order forms (36.1%); automatic stop of delivery (43.5%); analysis of antibiotic consumption data (96.2%); and analysis of microbial resistance data (89.8%).

These data demonstrate a well-developed structure of AMTs in Belgian hospitals and the broad range of services provided. Technical and financial support by healthcare authorities was key to the extensive implementation of antimicrobial stewardship programmes across the national hospital care system.

In the present study we analysed 26 XDR-TB clinical isolates. The gyrA, tlyA and rrs genes were screened for mutations that could be responsible for resistance to fluoroquinolones and second-line injectable drugs. Moreover, the strains under analysis were also genotyped by MIRU-VNTR (‘mycobacterial interspersed repetitive unit-variable number of tandem repeats’).

The mutational analysis identified the most frequent mutations in the resistance-associated genes: S91P in gyrA (42.3%); A1401G in rrs (30.8%); and Ins755GT in tlyA (42.3%). The occurrence of mutations in rrs was associated with the non-occurrence of mutations in tlyA. The genotypic analysis revealed that the strains were highly clonal, belonging to one of two MIRU-VNTR clusters, with the largest belonging to the Lisboa family. Association between mutations in gyrA and rrs or tlyA was verified.

The association of specific mutations highlighted the strains’ high clonality and indicates recent XDR-TB transmission. In addition, the identification of the most frequent resistance-associated mutations will be invaluable in applying XDR-TB molecular detection tests in the region in the near future.

Biocide susceptibilities, efflux and in vitro inactivation profiles were monitored in the presence/absence of efflux pump inhibitors. The RND transporters encoded by adeB and adeJ were detected by PCR; null mutants were constructed in the native host. Expression of adeB and adeJ in clinical isolates was assayed by semi-quantitative RT–PCR.

Susceptibility testing and phenotypic assays demonstrated the role of active efflux in mediating decreased susceptibility to biocides. Inactivation of either the adeB or adeJ transporter gene led to increased susceptibility to biocides. RT–PCR analysis exhibited increased adeB and adeJ expression in clinical isolates.

This is the first study demonstrating the role of efflux pumps in mediating decreased susceptibility to disinfectants and other chemical substrates in A. baumannii.

MICs were determined by broth microdilution and Etest. The presence of carbapenemase-encoding genes was investigated by PCR. Molecular epidemiology was performed by repetitive sequence-based PCR (rep-PCR; DiversiLab), sequence-type multiplex PCR and PFGE.

Imipenem non-susceptibility was associated with ISAba1 upstream of the intrinsic bla_OXA-51-like or the acquired carbapenemase bla_OXA-23-like, bla_OXA-40-like or bla_OXA-58-like. Isolates were grouped into eight distinct clusters including European clones I, II and III. European clone II was the largest (246 isolates) and most widespread group (USA, pan-Europe, Israel, Asia, Australia and South Africa).

The global dissemination of eight carbapenem-resistant lineages illustrates the success this organism has had in epidemic spread. The acquired OXA enzymes are widely distributed but are not the sole carbapenem resistance determinant in A. baumannii.

In silico analysis revealed the presence of a gene encoding a Qnr-like protein that shares 80% amino acid identity with QnrB1 in the S. marcescens strain Db11. Fragments carrying the coding region and the upstream non-coding sequences of eight clinical isolates were cloned and expressed in E. coli. MIC values of quinolones were determined. RT–PCR was used to study expression of these genes in their natural host. Southern hybridization was used to explore the presence of the gene in the genus Serratia.

Recombinant plasmids encoding SmaQnr reduced susceptibility to fluoroquinolones and nalidixic acid in both E. coli ATCC 25922 and DH10B. Sequences upstream of these genes contain a LexA box. Conventional RT–PCR showed transcription of the analysed Smaqnr genes in their natural hosts. Southern blot analysis suggests the presence of similar genes in several species of the genus Serratia.

SmaQnr conferred a reduced susceptibility phenotype against fluoroquinolones in E. coli. These data provide evidence of its possible role in quinolone resistance in S. marcescens. This Gram-negative species may constitute a reservoir for qnr-like quinolone resistance genes.

Mechanisms of carbapenem resistance were investigated in seven selected isolates (isolated between 2006 and 2008) belonging to the epidemic clone. Isolates underwent MIC testing, and were examined for the presence of KPC, Tn4401, class I integron elements and additional antibiotic resistance genes. Plasmids were analysed by transformation, transconjugation, restriction mapping, curing and complementation experiments. Outer membrane protein (OMP) analysis was performed.

OMP analysis did not reveal loss of porins. KPC-3-producing K. pneumoniae isolates possessed various plasmids but all harboured a common self-transmissible 105 kb plasmid, termed pKpQIL, encoding bla_TEM-1 and bla_KPC-3. Curing of pKpQIL led to a complete loss of resistance to cephalosporins and carbapenems, proving its crucial role in carbapenem resistance. Transformation of plasmid pKpQIL into the cured Klebsiella strain resulted in full reconstitution of carbapenem resistance. The presence of all Tn4401 transposon elements located upstream of the KPC-3 gene was detected by PCR and sequencing. pKpQIL lacked additional antibiotic resistance genes.

Our findings demonstrate the presence of pKpQIL, a 105 kb KPC-3- and TEM-1-encoding plasmid, in the XDR K. pneumoniae epidemic strain in Israel. pKpQIL is unique and appears consistently in all isolates of this clone over the years. The extensive β-lactam resistance phenotype of this clone is primarily mediated by this single self-transmissible plasmid.

Twenty-eight K. pneumoniae clinical isolates with reduced susceptibility to carbapenems were evaluated. Antimicrobial susceptibility and molecular typing were performed by agar dilution and PFGE, respectively. The MHT was performed using both standard and high inoculum of test organisms. Imipenem hydrolysis was investigated by spectrophotometric assays and carbapenemase-encoding genes were identified by PCR and amplicon sequencing. Porin loss was investigated by both PCR and SDS–PAGE.

Susceptibility rates for imipenem, meropenem and ertapenem were 93%, 57% and 11%, respectively. The PFGE analysis showed seven unrelated genotypes. By testing standard inoculum and ertapenem or meropenem discs, 25% (n = 7) and 21% (n = 6) of the isolates were classified as carbapenemase producers, respectively. When a higher inoculum was employed, these rates increased to 54% (n = 15) and 43% (n = 12), respectively. No imipenem hydrolysis was detected. PCRs identified bla_CTX-M in 27 (96%) isolates, of which 2 isolates also carried bla_GES-1. SDS–PAGE and PCR assays revealed that all isolates had lost at least one outer membrane protein, except for a single isolate that was found to express both OmpK35 and OmpK36.

False detection of carbapenemase production was observed by the MHT possibly as a result of extended-spectrum β-lactamase (ESBL) production coupled with porin loss as reported before. Clinical laboratories must be aware of this fact, especially in geographical areas where ESBL-producing isolates are highly prevalent.

BLI-489 was used at a constant concentration of 4 mg/L. Spontaneous mutation frequency was measured on piperacillin/BLI-489-containing agar plates. Five β-lactamase-producing strains were exposed to a serial dilution of piperacillin/BLI-489, and the highest concentration allowing growth was used to inoculate subsequent serial passage for 10 days. Mutation stability was monitored in drug-free medium for 10 days.

Escherichia coli (OXA-3, OXA-7, ACT-1, SHV-1 or none), Salmonella enterica serovar Typhimurium (CTX-M-5), Klebsiella pneumoniae (SHV-1 and SHV-5) and Enterobacter cloacae (AmpC) had a spontaneous mutation frequency of ≤1.0 x 10^–9. Two AmpC-producing Pseudomonas aeruginosa strains had a mutation frequency of 6.52 x 10^–6 and 1.0 x 10^–7; a β-lactamase-negative P. aeruginosa strain had a mutation frequency of 2.68 x 10^–8. The mutant prevention concentration (MPC) was ≤32 mg/L. During serial passages, the MIC increased 64- and 128-fold for S. enterica serovar Typhimurium (CTX-M-5) and E. cloacae (AmpC), respectively, to ≥512 mg/L. The MIC reverted to ≤64 mg/L after serial passages in drug-free medium. The MICs increased only 4-fold for K. pneumoniae (SHV-1 and SHV-5), E. coli (OXA-3) and E. coli (SHV-1).

Piperacillin/BLI-489 demonstrated a low probability of spontaneous resistance development in vitro for all of the strains tested with the exception of P. aeruginosa. The MPC value for all strains was ≤32 mg/L. Resistance developed during serial passage for two of the five strains tested; however, this resistance phenotype was unstable as MIC values reverted to ≤64 mg/L after propagation in drug-free medium.

The antimicrobial activity of a commercially available silver-processed external ventricular drain catheter was evaluated against Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Propionibacterium acnes. Non-impregnated catheters were used as controls. Two assays were performed: (i) testing the ability of the catheter to kill 100% of the attached bacteria (tK100); and (ii) in vitro challenge to determine the ability to prevent colonization under flow conditions. High and low inocula (10⁴ and 10⁷ cfu/mL) were used. Silver-processed and control catheters were examined by scanning electron microscopy and focused ion beam scanning electron microscopy; electron back-scatter and energy-dispersive X-ray analyses were used to investigate the distribution of silver within the processed catheter.

The silver-processed catheters were not able to kill any of the bacteria tested in the tK100 assay at high inoculum. At low inoculum S. epidermidis was eradicated and some activity was seen against E. coli but without complete eradication. MRSA was also not eradicated even at low inoculum. The in vitro challenge test showed no prevention of colonization for any of the strains. Silver particles were shown to be >500 nm in size.

The commercial silver-impregnated catheter was not able to eradicate MRSA or E. coli and while it showed activity against S. epidermidis in one assay it was unable to prevent colonization in vitro under in-flow conditions. This is consistent with clinical studies on silver-processed catheters.

MICs were determined on Mueller–Hinton agar supplemented with 2,2' bipyridyl to induce iron transport; comparators were aztreonam, imipenem, meropenem and piperacillin/tazobactam.

BAL30072 was strikingly active against Acinetobacter baumannii, with 73% of 200 carbapenemase-producing isolates, most of them belonging to the UK-dominant OXA-23 clone 1 and SE clone lineages, susceptible at 1 mg/L and 89% at 8 mg/L. Resistance nevertheless was seen in a few representatives of these clones and appeared commoner among isolates representing other A. baumannii clones. Sixty-eight per cent of 50 B. cepacia complex isolates from cystic fibrosis (CF) were susceptible to BAL30072 at 1 mg/L and 78% at 8 mg/L, compared with only 22% susceptible to aztreonam at 8 mg/L. Activity against P. aeruginosa was good, though less dramatic, with 36% of 50 (mostly multiresistant) CF isolates susceptible at 8 mg/L, compared with 12% susceptible to aztreonam at 8 mg/L. BAL30072 was active against 11/19 metallo-β-lactamase-producing P. aeruginosa at 8 mg/L compared with 3/19 for aztreonam (12/19 versus 8/19 at 16 mg/L). Studies on P. aeruginosa mutants, isolates and transconjugants showed that BAL30072 was affected by efflux, AmpC and by a few uncommon acquired β-lactamases, including some extended-spectrum OXA types and PER-1.

BAL30072 displayed impressive activity against many carbapenemase-producing A. baumannii, particularly against the two clones most prevalent in the UK, and also against B. cepacia complex isolates from CF; it was more active than aztreonam against P. aeruginosa.

Ten C. albicans bloodstream isolates used in this study were collected from intensive care patients admitted to the Vienna University Hospital between 2006 and 2007. Chequerboard tests were employed to determine the efficacy of the antifungal combinations amphotericin B/caspofungin and amphotericin B/posaconazole against both planktonic cells and biofilms. C. albicans biofilms were prepared using the static microtitre plate model. The activity of antifungal combination therapy was determined by visual reading for planktonic cells and using the XTT assay for biofilms.

For Candida biofilms the median MIC was 4 mg/L for amphotericin B and caspofungin, and >256 mg/L for posaconazole. The combination amphotericin B/posaconazole yielded synergism [fractional inhibitory concentration index (FICI) <0.26], whereas amphotericin B/caspofungin yielded indifferent interaction only (FICI 0.75–1.25) against all isolates when grown in biofilms. Under planktonic conditions, synergism was demonstrable for the combination amphotericin B/caspofungin against 4 of the 10 isolates, whereas the combination of caspofungin/posaconazole was indifferent against all tested isolates.

We showed that MICs for planktonic and biofilm forms of C. albicans were much lower when treated with an antifungal combination than when treated with single agents. The combination of amphotericin B/posaconazole yielded synergism against Candida biofilms, whereas amphotericin B/caspofungin yielded indifferent interaction.

HAE culture and ex vivo human bronchi were used to evaluate the sialic acid removal and regeneration efficiency and IFV inhibition after various DAS181 treatment levels and regimens.

DAS181 effectively desialylates HAE cultures and ex vivo bronchi tissues and therefore potently inhibits replication of different IFV strains. The treatment effect of DAS181 occurs immediately upon application to the epithelial surface and is unaffected by the respiratory mucus. In both HAE and human bronchial tissue, the inhibitory effect of DAS181 treatment lasts for at least 2 days. Approximately 80% epithelial surface desialylation and significant anti-IFV efficacy can be achieved at topical concentrations of DAS181 in the range of 5–10 µg/cm² when applied once daily. An additional treatment or a higher loading dose of DAS181 on the first day provides significant additional treatment benefit. Comparing the effect of DAS181 versus its two analogues, DAS180 and DAS185, has confirmed that sialidase function is critical for DAS181, and the cell-binding domain (amphiregulin tag) prolongs DAS181 retention and potentiates its function.

These results provide valuable insights into DAS181 treatment dose and potential regimens in the clinical setting.

The in vitro antifungal effects of the volatiles were assayed by a variety of methods. In vitro mammalian cell toxicity of the oil and the oil volatiles was also determined prior to animal testing. Efficacy of the volatiles in vivo was assessed using a murine model.

L. petersonii oil volatiles were found to be potent inhibitors of fungal growth in vitro, with fungicidal activity displayed following short exposure times (≤1 h). No significant mammalian cell toxicity was found to be associated with the volatiles. In the absence of treatment, Aspergillus fumigatus infection of animals resulted in an increase in inflammatory cell counts and high fungal burden within the lung tissue. Chitin levels in treated animals were significantly reduced compared with control animals. No viable fungi could be recovered from animals that had completed the treatment regimen.

The significant reduction in fungal burden in the lungs of infected animals by the volatiles of L. petersonii oil was larger than that reported for conventional antifungal drugs of choice.

In vitro MIC and minimum fungicidal concentration (MFC) values of the iron chelator, deferasirox, for Aspergillus fumigatus were determined by microdilution assay. In addition, we studied the efficacy of deferasirox alone or combined with LAmB in treating immunocompromised mice infected with A. fumigatus via inhalation.

Deferasirox was cidal in vitro against A. fumigatus, with an MIC and MFC of 25 and 50 mg/L, respectively. Deferasirox monotherapy modestly prolonged survival of mice with IPA. Combination deferasirox + LAmB therapy synergistically improved survival and reduced lung fungal burden compared with either monotherapy alone.

Iron chelation therapy with deferasirox alone or in combination with LAmB is effective in treating experimental IPA. Further study of deferasirox is warranted as adjunctive therapy for IPA infections.

We reviewed the in vitro susceptibilities (M27-A3 CLSI method) of 650 Candida spp. associated with invasive candidiasis episodes in 582 hospitalized cancer patients (2005–08).

We identified seven caspofungin-non-susceptible Candida strains (three Candida tropicalis, two Candida glabrata and two Candida albicans) from 650 Candida isolates (1%). C. tropicalis (three out of seven) was the most common non-susceptible species isolated. All patients responded to a change of antifungal therapy. Further surveillance should focus on the potential broader emergence of echinocandin resistance, as the clinical use of this antifungal class continues to expand in cancer patients.

Fungiscope^TM—A Global Rare Fungal Infection Registry is an international university-based case registry that collects data of patients with rare IFD, using a web-based electronic case form at www.fungiscope.net.

Forty-one patients with invasive zygomycosis from central Europe and Asia were registered. The most common underlying conditions were malignancies (n = 26; 63.4%), diabetes mellitus (n = 7; 17.1%) and solid organ transplantation (n = 4; 9.8%). Diagnosis was made by culture in 28 patients (68.3%) and by histology in 26 patients (63.4%). The main sites of infection were the lungs (n = 24; 58.5%), soft tissues (n = 8; 19.5%), rhino-sinu-orbital region (n = 8; 19.5%) and brain (n = 6; 14.6%). Disseminated infection of more than one non-contiguous site was seen in six patients (14.6%). Mycocladus corymbifer was the most frequently identified species (n = 10, 24.4%). A favourable response was observed in 23 patients (56.1%). Overall survival was 51.2% (n = 21). At diagnosis, four patients (9.8%) were on continuous antifungal prophylaxis with itraconazole (n = 1; 2.4%) or posaconazole (n = 3; 7.3%). Initial targeted treatment with activity against zygomycetes was administered to 34 patients (82.9%). Liposomal amphotericin B was associated with improved response (P = 0.012) and survival rates (P = 0.004).

Pathogen distribution and, consequently, drug susceptibility seem to vary across different geographic regions. Furthermore, protection from invasive zygomycosis for patients on posaconazole prophylaxis is not absolute. Our findings indicate that the use of liposomal amphotericin B as first-line treatment for patients diagnosed with zygomycoses merits further investigation, preferably in the form of a clinical trial.

We carried out a retrospective study in 109 patients. Chemokines were measured using Multiplex kits using a Luminex 100 Analyzer. Logistic regression was used to evaluate the association between plasma chemokine levels before HCV therapy and virological response at weeks 48 and 72 after starting HCV therapy.

Fifty-seven patients out of 103 achieved end of treatment virological response (ETR). In patients achieving ETR, the baseline levels of eotaxin, MCP-1 and MCP-3 were higher than non-responder (NR) patients. Similarly, 51 patients out of 106 achieved sustained virological response (SVR). In patients achieving SVR, the baseline levels of eotaxin and MCP-1 were higher than in NR patients. Plasma levels of eotaxin, MCP-1 and MCP-3 had a significant positive association with ETR, as well as eotaxin and MCP-1 with SVR. However, after stepwise multivariate logistic regression, eotaxin was the only chemokine selected capable of predicting ETR and SVR with odds ratio (OR) of 1.016 (95% CI: 1.004–1.029) and 1.015 (95% CI: 1.002–1.027) for ETR and SVR, respectively.

Our preliminary data suggest that plasma eotaxin levels prior to HCV antiviral therapy may be useful in predicting virological response to HCV treatment with IFN- + ribavirin in HIV/HCV co-infected patients. Further experimental research is necessary to corroborate this hypothesis.

HIV was analysed by PG and CG (771 baseline and 657 VF clones) from subjects with VF (confirmed HIV RNA ≥ 400 copies/mL at 24–48 weeks).

Fourteen of 123 subjects (11%) met VF criteria; their median baseline HIV RNA was 5.4 log₁₀ copies/mL, and 4.0 log₁₀ copies/mL at VF. By baseline PG, 2/14 had HIV-1 with nucleoside reverse transcriptase inhibitor (NRTI) or non-NRTI mutations. By baseline CG, 9/14 had HIV-1 with NNRTI and/or NRTI mutations; 7/9 had study drug-associated mutations. By PG at VF, 10/14 had selected for resistance mutations [2, K65R; 1, M184V; and 7, thymidine analogue mutations (TAMs) ± M184V]. By CG at VF, for subjects with TAMs, T215F was more commonly detected (5/14 samples) than T215Y (2/14). For one subject who selected K65R at VF, both K65R-containing clones and TAM-containing clones (both T215A and T215F) were observed independently but not conjunctively in the same clone in a post-VF sample.

The majority of subjects with VF had major and minor mutations detected at VF; CG detected additional low-abundance variants at baseline and VF that could have influenced mutation selection pathways. Both PG and CG data suggest TAMs, not K65R selection, are the preferred resistance route, biased towards 215F selection. No HIV clone contained both K65R and T215F/Y mutations, suggesting in vivo antagonism between the two mutations. The once-daily zidovudine usage and high baseline viraemia may also have contributed to rapid selection of HIV with multiple mutations in VFs.

This study was nested within a large double-blind placebo-controlled study designed to determine if primary prophylaxis with fluconazole (200 mg three times per week) could reduce cryptococcal disease [CRYPTOPRO (ISRCTN 76481529)] in HIV-infected adults in rural south-western Uganda. Detailed pharmacokinetic studies were performed on 49 participants (22 on placebo and 27 on fluconazole) who had been on fluconazole or placebo with nevirapine for ≥4 weeks.

The geometric mean pre-dose concentrations of nevirapine were 3865 ng/mL [95% confidence interval (95% CI) 3452–4758 ng/mL] and 5141 ng/mL (95% CI 4760–6595 ng/mL) (P = 0.009) in the placebo and fluconazole arms, respectively. The change in the peak nevirapine concentration in plasma (C_max) was also higher in the fluconazole arm compared with the placebo arm [median 6546 (95% CI 6040–7974) versus 5126 (95% CI 4739–5773) ng/mL, P = 0.012]. Fluconazole increased the nevirapine area under the curve (AUC) from 0 to 8 h by 29% [geometric mean AUC_0–8 46 135 (95% CI 42 432–57 173) versus 35 871 (95% CI 32 808–41 372) ng·h/mL, P = 0.016]. In the larger cohort from which the participants were drawn, co-administration of fluconazole did not increase the risk of hepatotoxicity.

Fluconazole led to significant increases in nevirapine exposure, but was not associated with evidence of increased hepatotoxicity.

Genetic sequences of HIV integrase were obtained from INI-naive patients at two European clinics. The 39 amino acid changes at 29 integrase positions so far associated with INI resistance were examined according to HIV clade, prior antiretroviral exposure and duration of HIV infection.

Integrase sequences were obtained from 418 patients, 294 (70.3%) infected with clade B and 124 (29.7%) infected with non-B variants (predominantly CRF02, A, C and D). Overall, 40% of patients were antiretroviral experienced and 32.8% were recent seroconverters. The most prevalent INI resistance-associated mutations were V72I (63.9%), V201I (54.8%), T206S (25.4%), I203M (9.8%) and K156N (7.4%). Major INI resistance mutations at positions 66, 92, 143, 148 and 155 were not detected. The mean number of polymorphic sites was greater in non-B than in B variants (2.17 versus 1.59; P < 0.001), and in antiretroviral-experienced than in drug-naive patients (1.89 versus 1.68; P = 0.034), whereas no significant differences were seen comparing recent seroconverters and chronically infected persons.

Major INI resistance-associated mutations are very rare, if indeed ever present, in INI-naive patients. However, polymorphisms at positions which may influence the genetic barrier and/or drive the selection of specific INI resistance pathways are common, especially in HIV non-B subtypes.

A post hoc subgroup study was selected from a double-blind, placebo-controlled trial, enrolling 146 patients with dyspepsia or peptic ulcers never previously treated. Real-time PCR and Etest on bacterial culture for assessing clarithromycin resistance were performed. [¹³C]urea breath test (UBT), histology and rapid urease tests at entry and UBT after 4–8 weeks were used to assess infection and eradication. All patients received a 10 day therapy.

Prevalence of clarithromycin phenotypic resistance was significantly lower as compared with genotypic resistance (18.4% versus 37.6%, P < 0.001). A concordance between the two methods was present in 71.2% of cases. A significant difference in the eradication rate was seen between clarithromycin-susceptible and -resistant strains, when assessed with either Etest (92.4% versus 55.5%, P < 0.001) or a PCR-based method (94.5% versus 70.9%; P < 0.001). Of note, the eradication rate showed the lowest value (30.7%) when phenotypic bacterial resistance was genetically linked to the A2143G point mutation.

This study showed that: (i) there is a relevant discordance between the two methods; and (ii) phenotypic clarithromycin resistance markedly reduces H. pylori eradication when it is linked to a specific point mutation.

Episodes of ESBL-EC bacteraemia were compared with a susceptible control group in a 3 year prospective study. ESBL-EC strains were studied by PCR and isoelectric focusing, and molecular typing was performed by PFGE.

Out of 531 episodes of bacteraemia, 135 were caused by E. coli. Seventeen of these cases involved ESBL-EC-producing strains (12.6%). In the multivariate analysis, female gender [odds ratio (OR) 3.43; 95% confidence interval (CI) 1.03–11.4] and previous antibiotic therapy (OR 3.22; 95% CI 1.00–10.3) were found to be independent risk factors for ESBL acquisition. An analysis of ESBL-EC isolates revealed a polyclonal distribution with CTX-M predominance (59%). Patients with ESBL-EC bacteraemia were more likely to have received an inadequate empirical antibiotic therapy (65% versus 6%; P = 0.000), and the time to adequate therapy was longer in this group (0 versus 1.50 days; P = 0.000). The overall mortality rate was 22%, ranging from 20% to 35% (P = 0.20). Risk factors for mortality were solid tumour (OR 19.41; 95% CI 4.66–80.83), corticosteroid therapy (OR 3.04 95% CI 1.05–8.81) and intensive care unit admission (OR 248.24, 95% CI 18.49–3332.14). In neutropenic patients, ESBL-EC bacteraemia was associated with poorer outcome and a higher overall mortality rate (37.5% versus 6.5%; P = 0.01).

In our centre, ESBL-EC bacteraemia is frequent among cancer patients, especially in those exposed to antibiotic pressure. All ESBL-EC strains were unrelated and most of them carried a CTX-M group enzyme. Patients with ESBL-EC bacteraemia received inadequate empirical antibiotic therapy more frequently than patients carrying a susceptible strain, but significant differences in mortality could not be demonstrated.

All consecutive patients, >15 years old, admitted to a surgical intensive care unit (ICU) between September 2006 and January 2009 for a first episode of POP were included. Antibiotic susceptibilities of microorganisms recovered from blood cultures and peritoneal fluid were determined by disc diffusion. Amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, piperacillin/tazobactam, cefotaxime, ceftazidime, cefepime, imipenem, gentamicin, amikacin and ciprofloxacin were tested against Gram-negative bacteria, and oxacillin, amoxicillin, vancomycin, gentamicin and erythromycin were tested against aerobic Gram-positive bacteria. Results were reported as susceptible or resistant.

MDRB were isolated from 20/115 (17%) patients. In univariate analysis, use of antimicrobial therapy during the 3 months prior to hospitalization and a long duration between hospital admission or first operation and relaparotomy were significantly associated with MDRB recovery. In multivariate analysis, only antimicrobial treatment in the 3 months preceding hospitalization and duration between first operation and relaparotomy were independent risk factors for MDRB [odds ratio (OR) = 5.80, 95% confidence interval (95% CI) = 1.99–16.91 and OR = 1.10, 95% CI = 1.02–1.19, respectively]. No MDRB were found when the delay between the first operation and relaparotomy was <5 days. POP severity, non-surgical and surgical complications, hospital and ICU length of stay, and mortality were similar in patients with and without MDRB.

Our results suggest that broad-spectrum antibiotics should be used in ICU patients with POP who have received antimicrobial therapy in the 3 months prior to hospitalization, or with >5 days between the first operation and relaparotomy.

Five hundred and seventy-one UK consultant microbiologists were contacted via e-mail and asked to take part in an online survey, hosted at www.surveymonkey.com. Responses were collated by the website, downloaded and analysed in a Microsoft^® Excel (Microsoft Corporation) spreadsheet.

One hundred and eight respondents participated in the survey. Only 32.7% routinely measure MICs, mostly by Etest. Forty-two percent use vancomycin alone for removable-focus infections, whilst for infections of cardiac or orthopaedic origin, 49% would add rifampicin. Few respondents use daptomycin, linezolid or tigecycline empirically. Sixty-nine percent would use linezolid as a second-line agent, with only 19% opting for daptomycin. For an isolate with a vancomycin MIC of 4 mg/L, respondents would use daptomycin (81%) or linezolid (91%) in patients with a poor clinical response.

Vancomycin is the mainstay therapy for MRSA BSIs, even when MICs are not measured or raised, despite evidence of high failure rates. The use of newer agents frequently does not follow European or US licensed indications, may be inappropriate and may result in avoidable deaths.

The intervention was performed on a 14 year baseline of monthly data on trimethoprim resistance and consumption. A three-parameter mathematical model was used to analyse the intervention effect. The prerequisites for reversion of resistance (i.e. fitness cost, associated resistance and clonal composition) were studied on subsets of consecutively collected Escherichia coli from urinary tract infections.

The use of trimethoprim-containing drugs decreased by 85% during the intervention. A marginal but statistically significant effect on the increase in trimethoprim resistance was registered. There was no change in the clonal composition of E. coli and there was no measurable fitness cost associated with trimethoprim resistance in clinical isolates. The frequency of associated antibiotic resistances in trimethoprim-resistant isolates was high.

A lack of detectable fitness cost of trimethoprim resistance in vitro together with a strong co-selection of other antibiotics could explain the rather disappointing effect of the intervention. The result emphasizes the low possibility of reverting antibiotic resistance once established and the urgent need for the development of new antibacterial agents.