Isolation of Rhodococcus equi from the gastrointestinal contents of earthworms (family Megascolecidae).

Rhodococcus equi was isolated from the gastrointestinal contents of earthworms (family Megascolecidae) and their surrounding soil collected from pastures of two horse-breeding farms in Aomori Prefecture, outdoor pig pens, forest in Towada campus, orange groves and forest where wild boars (Sus scrofa) are established in Tanabe, Wakayama Prefecture. The number of R. equi in the lower gastrointestinal contents of 23 earthworms collected from our campus was significantly larger than that of the upper gastrointestinal content. The mean numbers of R. equi from the gastrointestinal contents of earthworms collected from the various places were 2·3-fold to 39·7-fold more than those of the surrounding soil samples. In all, 1771 isolates from the earthworms and 489 isolates from the soil samples were tested for the presence of vapA and vapB genes using polymerase chain reaction. At the horse-breeding farm N, 9 of the 109 isolates (8·3%) from the earthworms and 7 of the 106 isolates (6·6%) from the soil samples were positive for the vapA gene. At the University's forest, one of the 250 isolates (0·4%) from the gastrointestinal contents of the earthworm was positive for the vapB gene. These results revealed that R. equi can be found in significant quantities in the gastrointestinal contents of earthworms, suggesting that they act as an accumulator of R. equi in the soil environment and as a source or reservoir of animal infection.

Virulence plasmids in clinical isolates of Rhodococcus equi from sick foals in the Netherlands.

Clinical samples from 123 foals with suspected rhodococcosis submitted to the Veterinary Microbiological Diagnostic Centre of the Faculty of Veterinary Medicine between 1993 and 2006 were tested for the presence of the virulence gene vapA. Of the 123 samples, 120 were vapA-positive and 3 vapA-negative Rhodococcus equi were isolated. The 120 vapA-positive R. equi were isolated from 70 tracheal wash, 19 lung tissues, 7 lymph nodes, 6 synovial fluids, 13 abscesses or pus and single isolates from the uterus, gut, cerebrospinal fluid, abdomen fluid and faeces. Of the 120 isolates, 46 were from Dutch warmblood horses, 23 from Friesian horses, 14 from Trotters, 4 from Holsteiners, 3 from Arab breed, 2 from ponies, 1 from a Welsh pony and 27 from undefined breed horses. Using plasmid profile analysis of the 120 isolates, 117 isolates contained the 85-kb type I plasmid, 2 contained the 87-kb type I plasmid and 1 contained the novel 52-kb non-mobilizable virulence plasmid reported recently. These results showed that the virulent R. equi strains harbouring a virulence plasmid of 85-kb type I or 87-kb type I, which have been detected in clinical isolates from five European countries, are widespread in the Netherlands. This is the first report of plasmid types of clinical R. equi isolates in the Netherlands.

Reinvestigation of the virulence of Rhodococcus equi isolates from patients with and without AIDS.

Rhodococcus equi emerged as a zoonotic pathogen of human immunodeficiency virus-infected patients over the last three decades. Two virulence plasmid types of R. equi, pVAPA and pVAPB associated with equine and porcine isolates, have been recognized, and more recently, pVAPN, a novel host-associated virulence plasmid in R. equi, was found in bovine and caprine isolates. We reinvestigated 39 previously reported isolates of R. equi from patients with and without acquired immunodeficiency syndrome (AIDS) by detecting vapA, vapB and vapN using PCR and plasmid profiling. After excluding one isolate that could not be cultured from frozen storage, eight isolates carried a virulence plasmid encoding vapA (pVAPA), 10 carried a virulence plasmid encoding vapB (pVAPB), seven carried a virulence plasmid encoding vapN (pVAPN) and 13 were negative for those genes. Of the 29 isolates from patients with AIDS, 7, 10 and 5 harboured pVAPA, pVAPB and pVAPN respectively. Among nine isolates from patients without AIDS, one and two harboured pVAPA and pVAPN respectively. This study demonstrated that pVAPN-positive R. equi existed in human isolates before 1994 and reaffirmed that equine-associated pVAPA-positive, porcine-associated pVAPB-positive and bovine- or caprine-associated pVAPN-positive R. equi are widely spread globally. Because domestic animals might be major sources of human infection, further research is needed to reveal the prevalence of pVAPN-positive R. equi infection in cattle and goats.

Fouling potential of lipopolysaccharides released at low temperatures in MBRs.

Sludge filterability in membrane bioreactors (MBRs) fluctuates and affects membrane fouling. Therefore, understanding the reasons for the fluctuations of sludge filterability is important for the efficient operation of MBRs. In this study, a pilot-scale MBR treating municipal wastewater was operated for about 600 days and the variations in sludge filterability were continuously monitored by batch-filtration experiments using the same membranes as in the MBR. To investigate the reasons for the deterioration of sludge filterability, constituents in sludge supernatant were intensively monitored, and the correlations with sludge filterability were determined. The concentration of lipopolysaccharides (LPS) in sludge supernatant exhibited significantly higher correlation with sludge filterability than did conventional indexes (i.e. polysaccharides and proteins). Size fractions affecting MBR sludge filterability were also investigated, and it was suggested that colloidal LPS deteriorated MBR sludge filterability. Based on the long-term operation of the MBR, increase in colloidal LPS under low temperatures of the mixed liquor suspension was a key factor in the deterioration of sludge filterability. The impact of LPS increasing under low temperatures should be investigated by operating bench-scale MBRs fed with synthetic wastewater in controlled conditions.

Efficacy and safety of 6 or 8 weeks of simeprevir, daclatasvir, sofosbuvir for HCV genotype 1 infection.

The phase 2, open-label ACCORDION (ClinicalTrials.gov: NCT02349048) study investigated the efficacy, safety and pharmacokinetics of a 6- or 8-week regimen of simeprevir, daclatasvir and sofosbuvir in treatment-naïve patients with chronic hepatitis C virus (HCV) genotype (GT) 1 infection and either early-stage fibrosis or compensated cirrhosis. Patients were assigned to treatment groups according to their fibrosis stage. Early-stage fibrosis: simeprevir 150 mg, daclatasvir 60 mg, sofosbuvir 400 mg once daily for 6 weeks; compensated cirrhosis: same regimen for 8 weeks. The primary endpoint was sustained virologic response 12 weeks after the end of treatment (SVR12). Safety, tolerability and pharmacokinetics of simeprevir, daclatasvir and sofosbuvir were investigated. Sixty-eight patients were treated (6-week group: n = 59; 8-week group: n = 9). SVR12 was achieved by 86.4% (51/59) of patients with early-stage fibrosis and by 100% (9/9) of patients with cirrhosis. The main reason for not achieving SVR12 in the 6-week group was viral relapse (11.9%; 7/59). One patient had on-treatment failure due to an early withdrawal (lost to follow-up due to incarceration). One patient with SVR12 in the 6-week group had a late viral relapse at post-treatment week 24. No clinically significant drug-drug interactions were observed. Adverse events were reported in 63.2% of patients (43/68) and were mainly grade 1/2. None of these led to treatment discontinuation. The 3 direct-acting antiviral regimens of simeprevir, daclatasvir and sofosbuvir were safe and well tolerated in treatment-naïve, HCV GT1-infected patients with early-stage fibrosis or compensated cirrhosis.

Simeprevir, daclatasvir and sofosbuvir for hepatitis C virus-infected patients with decompensated liver disease.

Approximately three million individuals in the United States are chronically infected with hepatitis C virus (HCV). Chronic HCV infection may lead to the development of compensated as well as decompensated liver cirrhosis. The Phase II IMPACT study was conducted in HCV genotype 1- or 4-infected cirrhotic patients with portal hypertension or decompensated liver disease and assessed for the first time the combination of the three direct-acting antivirals simeprevir, daclatasvir and sofosbuvir. Treatment-naïve or treatment-experienced adults with Child-Pugh (CP) score <7 (CP A) and evidence of portal hypertension, or CP score 7-9 (CP B), received 12 weeks of simeprevir 150 mg, daclatasvir 60 mg and sofosbuvir 400 mg, once daily. The primary efficacy endpoint was sustained virologic response 12 weeks after end of treatment (SVR12). Pharmacokinetics and safety were also assessed. Overall, 40 patients were enrolled (CP A: 19; CP B: 21). All 40 patients achieved SVR12. At week 8, the mean pharmacokinetic exposure to simeprevir, sofosbuvir, daclatasvir and GS-331007 (sofosbuvir metabolite) was 2.2-, 1.5-, 1.2- and 1.2-fold higher in patients with CP B than CP A, respectively. Grade 1/2 adverse events (AEs) occurred in 26 of 40 (65%) patients. One CP B patient had a Grade 3 AE (gastrointestinal haemorrhage), which was reported as a serious AE but not considered related to study drugs. Treatment for 12 weeks with simeprevir, daclatasvir and sofosbuvir was generally safe and well tolerated, and resulted in 100% of cirrhotic patients with portal hypertension or decompensated liver disease achieving SVR12.

Pharmacokinetics of once-daily darunavir/ritonavir in HIV-1-infected pregnant women.

OBJECTIVES: HIV antiretroviral therapy during pregnancy is recommended to reduce the risk of mother-to-child transmission and for maternal care. Physiological changes during pregnancy can affect pharmacokinetics. The impact of pregnancy was evaluated for once-daily (qd) darunavir/ritonavir. METHODS: HIV-1-infected pregnant women on an antiretroviral regimen that includes darunavir were enrolled in the study and further treated with darunavir/ritonavir 800/100 mg qd. Plasma concentrations were assessed over 24 h during the second and third trimesters and postpartum using a validated high-performance liquid chromatography tandem mass spectrometry assay for total darunavir and ritonavir, and using (14) C-darunavir-fortified plasma for unbound darunavir. Pharmacokinetic parameters were derived using noncompartmental analysis. Safety and antiviral response were assessed at all visits. RESULTS: Data were available for 16 women. The area under the plasma concentration-time curve from 0 to 24 h (AUC24h ) for total darunavir was 34-35% lower during pregnancy vs. postpartum. Unbound darunavir AUC24h was 20-24% lower during pregnancy vs. postpartum. The minimum plasma concentration of total and unbound darunavir was 32-50% and 13-38% lower, respectively, during pregnancy vs. postpartum. The antiviral response (< 50 HIV-1 RNA copies/mL) was 59% at baseline and increased to 87-100% during the trial; the CD4 count increased over time. One serious adverse event (gestational diabetes) was judged as possibly related to study medication. All 16 infants born to women remaining in the study at delivery were HIV-1 negative (two were premature). CONCLUSIONS: Total darunavir exposure decreased during pregnancy, but the decrease was less for unbound (active) darunavir. These changes are not considered clinically relevant. Darunavir/ritonavir 800/100 mg qd may therefore be a treatment option for HIV-1-infected pregnant women.

The Intelence aNd pRezista Once A Day Study (INROADS): a multicentre, single-arm, open-label study of etravirine and darunavir/ritonavir as dual therapy in HIV-1-infected early treatment-experienced subjects.

OBJECTIVES: Following antiretroviral therapy failure, patients are often treated with a three-drug regimen that includes two nucleoside/tide reverse transcriptase inhibitors [N(t)RTIs]. An alternative two-drug nucleoside-sparing regimen may decrease the pill burden and drug toxicities associated with the use of N(t)RTIs. The Intelence aNd pRezista Once A Day Study (INROADS; NCT01199939) evaluated the nucleoside-sparing regimen of etravirine 400 mg with darunavir/ritonavir 800/100 mg once-daily in HIV-1-infected treatment-experienced subjects or treatment-naïve subjects with transmitted resistance. METHODS: In this exploratory phase 2b, single-arm, open-label, multicentre, 48-week study, the primary endpoint was the proportion of subjects who achieved HIV-1 RNA < 50 copies/mL at week 48 [confirmed virological response (CVR), non-virological failure (VF) censored]. Key secondary endpoints included assessments of changes from baseline to week 48 in viral load, immunological response, pharmacokinetics/pharmacodynamics, safety, tolerability, metabolic and bone markers and body fat. RESULTS: Forty-one of the 54 enrolled subjects completed the study. Adverse events (7%) and VF (7%) were the most common reasons for discontinuation. The week 48 CVR rate in the intent-to-treat (ITT) non-VF censored population was 89% (primary endpoint). Seven subjects experienced VF. Common adverse events were diarrhoea (15%), rash (15%) and upper respiratory tract infection (11%). Mild/moderate lipid elevations, minimal changes in limb fat distribution and bone mineral density and no clinically relevant changes in glucose metabolism were observed. CONCLUSIONS: Etravirine 400 mg and darunavir/ritonavir 800/100 mg as a two-drug once-daily regimen in treatment-experienced subjects or treatment-naïve subjects with transmitted resistance was virologically efficacious and well tolerated.

VapB type 8 plasmids in Rhodococcus equi isolated from the small intestine of pigs and comparison of selective culture media.

UNLABELLED: The virulence-plasmid profile of Rhodococcus equi strains isolated from Suidae and humans is similar. Recent evidence suggests that the consumption of pork products contaminated with faeces might be a potential source of R. equi infections in humans, mainly to patients with rhodococcosis without history of contact with pigs or pig farms. This study investigated the virulence-associated genes (vapA and vapB) and plasmid profiles of R. equi among the 150 samples of small intestinal content obtained from slaughtered pigs. In addition, all samples were subjected to microbiological culture in conventional sheep blood agar and CAZ-NB, TCP and TVP selective media. A total of 40 (26·7%) of the samples recovered R. equi, with two samples recovering isolates harbouring the VapB type 8 plasmid. Among the 150 pigs sampled herein, CAZ-NB was considered the best selective medium for the isolation of R. equi from faeces. Our results provide evidence that the contamination of slaughtered pig carcasses with pathogenic R. equi might occur through faeces, representing a public health concern. Furthermore, this study is the first description of R. equi strains carrying the VapB plasmid in the gut of pigs. SIGNIFICANCE AND IMPACT OF THE STUDY: Intermediately virulent (VapB) is a common plasmid-type harboured by R. equi isolated from pigs and humans with AIDS. Curiously, humans with rhodococcosis usually have no history of contact with pigs or pig farms. Virulence-plasmid profile of 40 R. equi isolated among 150 small intestine content samples from pigs revelled two carrying isolates with the VapB type-8 plasmids. Moreover, comparison of three selective culture media shows that CAZ-NB was the best. Our results provide evidence that contamination of slaughtered pig carcasses with pathogenic R. equi might occur through faeces, representing a public health concern. Furthermore, R. equi carrying VapB type-8 plasmids types are described for the first time in the gut of the pig.

Total and unbound darunavir pharmacokinetics in pregnant women infected with HIV-1: results of a study of darunavir/ritonavir 600/100 mg administered twice daily.

OBJECTIVES: Antiretroviral therapy during pregnancy is recommended to reduce the risk of mother-to-child transmission of HIV and for maternal care management. Physiological changes during pregnancy can affect pharmacokinetics, potentially altering pharmacological activity. We therefore evaluated the pharmacokinetics of twice-daily (bid) darunavir in HIV-1-infected pregnant women. METHODS: HIV-1-infected pregnant women receiving an antiretroviral regimen containing darunavir/ritonavir 600/100 mg bid were enrolled in this study. Total and unbound darunavir and total ritonavir plasma concentrations were obtained over 12 h during the second and third trimesters and postpartum. Total darunavir and ritonavir plasma concentrations were determined using a validated high-performance liquid chromatography tandem mass spectrometry assay and unbound darunavir was determined using (14) C-darunavir-fortified plasma. Pharmacokinetic parameters were derived using noncompartmental analysis. RESULTS: Data were available for 14 women. The area under the plasma concentration-time curve from 0 to 12 h (AUC12h) for total darunavir was 17-24% lower during pregnancy than postpartum. The AUC12h for unbound darunavir was minimally reduced during pregnancy vs. postpartum. The minimum plasma concentration (Cmin) of total and unbound darunavir was on average 43-86% and 10-14% higher, respectively, during pregnancy vs. postpartum. The antiviral response (< 50 HIV-1 RNA copies/mL) was 33% at baseline and increased to 73-90% during treatment; the percentage CD4 count increased over time. One serious adverse event was reported (increased transaminase). All 12 infants born to women remaining in the study at delivery were HIV-1-negative; four of these infants were premature. CONCLUSIONS: Total darunavir exposure decreased during pregnancy. No clinically relevant change in unbound (active) darunavir occurred during pregnancy, suggesting that no dose adjustment is required for darunavir/ritonavir 600/100 mg bid in pregnant women.

Etravirine in treatment-experienced, HIV-1-infected children and adolescents: 48-week safety, efficacy and resistance analysis of the phase II PIANO study.

OBJECTIVES: PIANO (Paediatric study of Intelence As an NNRTI Option; TMC125-C213; NCT00665847) assessed the safety/tolerability, antiviral activity and pharmacokinetics of etravirine plus an optimized background regimen (OBR) in treatment-experienced, HIV-1-infected children (≥ 6 to < 12 years) and adolescents (≥ 12 to < 18 years) over 48 weeks. METHODS: In a phase II, open-label, single-arm study, 101 treatment-experienced patients (41 children; 60 adolescents) with screening viral load (VL) ≥ 500 HIV-1 RNA copies/mL received etravirine 5.2 mg/kg (maximum dose 200 mg) twice a day (bid) plus OBR. RESULTS: Sixty-seven per cent of patients had previously used efavirenz or nevirapine. At week 48, the most common treatment-related grade ≥ 2 adverse event (AE) was rash (13%); 12% experienced grade 3 AEs. Only two grade 4 AEs occurred (both thrombocytopaenia, not etravirine related). At week 48, 56% of patients (68% children; 48% adolescents) achieved a virological response (VL<50 copies/mL; intent-to-treat, noncompleter=failure). Factors predictive of response were adherence > 95%, male sex, low baseline etravirine weighted genotypic score and high etravirine trough concentration (C0h ). Seventy-six patients (75%) completed the trial; most discontinuations occurred because of protocol noncompliance or AEs (8% each). Sixty-five per cent of patients were > 95% adherent by questionnaire and 39% by pill count. Forty-one patients experienced virological failure (VF; time-to-loss-of-virological-response non-VF-censored algorithm) (29 nonresponders; 12 rebounders). Of 30 patients with VF with paired baseline/endpoint genotypes, 18 (60%) developed nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations, most commonly Y181C. Mean etravirine area under the plasma concentration-time curve over 12 h (AUC0-12h ; 5216 ng h/mL) and C0h (346 ng/mL) were comparable to adult target values. CONCLUSIONS: Results with etravirine 5.2 mg/kg bid (with OBR) in this treatment-experienced paediatric population and etravirine 200 mg bid in treatment-experienced adults were comparable. Etravirine is an NNRTI option for treatment-experienced paediatric patients.

Pharmacokinetic interaction between etravirine or darunavir/ritonavir and artemether/lumefantrine in healthy volunteers: a two-panel, two-way, two-period, randomized trial.

OBJECTIVES: Etravirine is a substrate and inducer of cytochrome P450 (CYP) 3A and a substrate and inhibitor of CYP2C9 and CYPC2C19. Darunavir/ritonavir is a substrate and inhibitor of CYP3A. Artemether and lumefantrine are primarily metabolized by CYP3A; artemether is also metabolized to a lesser extent by CYP2B6, CYP2C9 and CYP2C19. Artemether has an active metabolite, dihydroartemisinin. The objective was to investigate pharmacokinetic interactions between darunavir/ritonavir or etravirine and arthemether/lumefrantrine. METHODS: This single-centre, randomized, two-way, two-period cross-over study included 33 healthy volunteers. In panel 1, 17 healthy volunteers received two treatments (A and B) in random order, with a washout period of 4 weeks between treatments: treatment A: artemether/lumefantrine 80/480 mg alone, in a 3-day course; treatment B: etravirine 200 mg twice a day (bid) for 21 days with artemether/lumefantrine 80/480 mg from day 8 (a 3-day treatment course). In panel 2, another 16 healthy volunteers received two treatments, similar to those in panel 1 but instead of etravirine, darunavir/ritonavir 600/100 mg bid was given. RESULTS: Overall, 28 of the 33 volunteers completed the study. Co-administration of etravirine reduced the area under the plasma concentration-time curve (AUC) of artemether [by 38%; 90% confidence interval (CI) 0.48-0.80], dihydroartemisinin (by 15%; 90% CI 0.75-0.97) and lumefantrine (by 13%; 90% CI 0.77-0.98) at steady state. Co-administration of darunavir/ritonavir reduced the AUC of artemether (by 16%; 90% CI 0.69-1.02) and dihydroartemisinin (by 18%; 90% CI 0.74-0.91) but increased lumefantrine (2.75-fold; 90% CI 2.46-3.08) at steady state. Co-administration of artemether/lumefantrine had no effect on etravirine, darunavir or ritonavir AUC. No drug-related serious adverse events were reported during the study. CONCLUSIONS: Co-administration of etravirine with artemether/lumefantrine may lower the antimalarial activity of artemether and should therefore be used with caution. Darunavir/ritonavir can be co-administered with artemether/lumefantrine without dose adjustment but should be used with caution.

Assessment of the steady-state pharmacokinetic interaction between etravirine administered as two different formulations and tenofovir disoproxil fumarate in healthy volunteers.

OBJECTIVE: Two open-label, randomized, cross-over trials in healthy volunteers were conducted to investigate the pharmacokinetic interaction between etravirine and tenofovir disoproxil fumarate. METHODS: Etravirine was administered as either 800 mg twice a day (bid) (phase II formulation in Study 1) or 200 mg bid (phase III formulation in Study 2) for 8 days followed by a 12 h pharmacokinetic evaluation. After a minimum of 14 days washout, tenofovir disoproxil fumarate 300 mg once a day was administered for 16 days. Volunteers were randomized to receive co-administration of etravirine with tenofovir disoproxil fumarate on either days 1-8 or days 9-16 followed by a 12 h pharmacokinetic evaluation for etravirine on day 8 or 16, respectively. Plasma and urine tenofovir concentrations were determined on days 8 and 16 over 24 h. RESULTS: The least square mean (LSM) ratio [90% confidence interval (CI)] for the area under the plasma concentration-time curve from 0 to 12 h (AUC(12 h)) for etravirine co-administered with tenofovir disoproxil fumarate vs. etravirine alone was 0.69 (0.61-0.79) and 0.81 (0.75-0.88) in Studies 1 and 2, respectively. The LSM ratio (90% CI) for the effect of etravirine on tenofovir AUC(24 h) was 1.16 (1.09-1.23) in Study 1 and 1.15 (1.09-1.21) in Study 2. CONCLUSIONS: These alterations are not considered clinically relevant for either drug and no dose adjustment is necessary when etravirine and tenofovir disoproxil fumarate are co-administered.

Impact of reverse transcriptase resistance on the efficacy of TMC125 (etravirine) with two nucleoside reverse transcriptase inhibitors in protease inhibitor-naïve, nonnucleoside reverse transcriptase inhibitor-experienced patients: study TMC125-C227.

OBJECTIVES: TMC125-C227, an exploratory phase II, randomized, controlled, open-label trial, compared the efficacy and safety of TMC125 (etravirine) with an investigator-selected protease inhibitor (PI) in nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant, protease inhibitor-naïve, HIV-1-infected patients. METHODS: Patients were randomized to TMC125 800 mg twice a day (bid) (phase II formulation; n=59) or the control PI (n=57), plus two nucleoside reverse transcriptase inhibitors (NRTIs). RESULTS: In an unplanned interim analysis, patients receiving TMC125 demonstrated suboptimal virological responses relative to the control PI. Therefore, trial enrolment was stopped prematurely and TMC125 treatment discontinued after a median of 14.3 weeks. In this first-line NNRTI-failure population, baseline NRTI and NNRTI resistance was high and reduced virological responses were observed relative to the control PI. No statistically significant relationship was observed between TMC125 exposure and virological response at week 12. TMC125 was better tolerated than a boosted PI for gastrointestinal-, lipid- and liver-related events. CONCLUSIONS: In a PI-naïve population, with baseline NRTI and NNRTI resistance and NRTI recycling, TMC125 was not as effective as first use of a PI. Therefore the use of TMC125 plus NRTIs alone may not be optimal in PI-naïve patients with first-line virological failure on an NNRTI-based regimen. Baseline two-class resistance, rather than pharmacokinetics or other factors, was the most likely reason for suboptimal responses.

Novel bovine-associated pVAPN plasmid type in Rhodococcus equi identified from lymph nodes of slaughtered cattle and lungs of people living with HIV/AIDS.

Rhodococcus equi is a well-recognized Gram-positive intracellular facultative bacterium that is opportunistic in nature, which causes pyogranulomatous infections in humans and multiple host animals. The pathogenicity of the microorganism has been attributed to the presence of plasmid-encoded virulence-associated proteins (Vap). To date, three host-associated virulence plasmid types of R. equi have been identified as follows: the circular pVAPA and pVAPB, related, respectively, to equine and porcine isolates, and a recently described linear pVAPN plasmid associated with bovine strains, although these three types are found in human isolates. Recent phylogenomic studies support the evidence that human R. equi infection is zoonotically acquired. Nevertheless, data regarding distribution and prevalence of the host-adapted virulence plasmid types of R. equi isolated from meat animals are scarce or unnoticed. Here, the three host-associated virulence plasmid types (pVAPA, pVAPB, and pVAPN) were investigated in 154 R. equi isolates recovered from lymph nodes of cattle with lymphadenitis (n = 31), faeces of cattle without enteric signs (n = 49), as well as different clinical specimens from human patients (n = 74). The analysis of virulence profile of 74 R. equi from humans revealed six (8.1%) isolates pVAPB (type 8), two (2.7%) pVAPN, and one (1.3%) pVAPB (type 11), all of which were from lung samples from people living with HIV/AIDS. From the lymph node samples of cattle, 41.9% (13 of 31) isolates revealed pVAPN type, whereas all isolates from faecal samples were negative for three host-associated types. Here, recently described bovine-associated pVAPN type was detected in R. equi isolates recovered from the lungs of people living with HIV/AIDS and lymph nodes from slaughtered cattle intended for human consumption; a finding that represents a public health concern, mainly in countries where undercooked or raw meat are traditionally consumed.

Genotypic characterization of VapA positive Rhodococcus equi in foals with pulmonary affection and their soil environment on a warmblood horse breeding farm in Germany.

Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) (P<0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive. R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive. The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.

Plasmid profiles of virulent Rhodococcus equi isolates from soil environment on horse-breeding farms in Hungary.

The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.

Model-Based Once-Daily Darunavir/Ritonavir Dosing Recommendations in Pediatric HIV-1-Infected Patients Aged ≥3 to <12 Years.

An existing population pharmacokinetic model of darunavir in adults was updated using pediatric data from two studies evaluating weight-based, once-daily dosing of darunavir/ritonavir (ARIEL, NCT00919854 and DIONE, NCT00915655). The model was then used to provide once-daily dosing recommendations for darunavir/ritonavir in pediatric patients aged ≥3 to <12 years. The final model comprised two compartments with first-order absorption and apparent clearance dependent on the concentration of α1-acid glycoprotein. The recommended darunavir/ritonavir once-daily dosing regimens in children aged ≥3 to <12 years are: 35/7 mg/kg from 10 to <15 kg, 600/100 mg from 15 to <30 kg, 675/100 mg from 30 to <40 kg, and 800/100 mg for ≥40 kg. These doses should result in exposures similar to the adult exposure after treatment with darunavir/ritonavir 800/100 mg once daily, while minimizing pill burden and allowing a switch from suspension to tablet(s) as early as possible.

Indinavir plasma protein binding in HIV-1-infected adults.

OBJECTIVE: To quantify unbound indinavir concentrations and characterize indinavir plasma protein binding in HIV-infected adults. DESIGN: Pharmacokinetic study in antiretroviral-naive, HIV-infected persons with CD4 T lymphocytes > 100 x 10(6) cells/L and HIV-RNA in plasma >5000 copies/ml at baseline who were participating in an open-label study of zidovudine, lamivudine and indinavir therapy. METHODS: Eight men underwent 8 h intensive pharmacokinetic studies for indinavir on two occasions 6 months apart. Unbound indinavir was separated by ultra-filtration, and unbound and total concentrations were quantified by a validated high-performance liquid chromatography method. RESULTS: Overall indinavir protein binding was 61+/-6%, with a range among the profiles of 54 to 70%. Indinavir binding was higher at the 8 h post-dose concentration compared with the 1 h post-dose concentration (66 versus 57%, P = 0.0006). CONCLUSIONS: The mean 61% protein binding for indinavir in these HIV-infected persons is similar to the in vitro report of 60%. However, the fraction bound was concentration-dependent, and considerable variability in binding was present among patients. Quantification of unbound protease inhibitor concentrations opens new avenues of research to advance our understanding of the pharmacologically-relevant moieties of antiretroviral agents and thereby the pharmacotherapy of HIV infection.