Mycothione reductase as a potential target in the fight against Mycobacterium abscessus infections.

While infections caused by Mycobacterium abscessus complex (MABC) are rising worldwide, the current treatment of these infections is far from ideal due to its numerous shortcomings thereby increasing the urge for novel drug targets. In this study, mycothione reductase (Mtr) was evaluated for its potential as a drug target for MABC infections since it is a key enzyme needed in the recycling of mycothiol, the main low-molecular-weight thiol protecting the bacteria against reactive oxygen species and other reactive intermediates. First, a Mab∆mtr mutant strain was generated, lacking mtr expression. Next, the in vitro sensitivity of Mab∆mtr to oxidative stress and antimycobacterial drugs was determined. Finally, we evaluated the intramacrophage survival and the virulence of Mab∆mtr in Galleria mellonella larvae. Mab∆mtr demonstrated a 39.5-fold reduction in IC90 when exposed to bedaquiline in vitro. Furthermore, the Mab∆mtr mutant showed a decreased ability to proliferate inside macrophages and larvae, suggesting that Mtr plays an important role during MABC infection. Altogether, these findings support the assumption of Mtr being a potential target for antimycobacterial drugs.IMPORTANCEMycobacterium abscessus complex (MABC) is a group of bacteria causing a serious public health problem worldwide due to its ability to cause progressive disease, its highly resistant profile against various antibiotics, and its lengthy treatment. Therefore, new drugs are needed to alleviate antibiotic resistance and reduce the length of the current treatment. A potential new target for new antibiotics is mycothione reductase (Mtr), an important enzyme belonging to a pathway that protects the bacteria against harmful conditions. Our research created a bacterium deficient of mtr by using advanced genetic techniques and demonstrated that mtr-deficient bacteria have a decreased ability to multiply during infection. Furthermore, we show evidence that currently used antibiotics combined with mtr deficiency can lead to a better treatment of MABC infection. Altogether, our results validate Mtr as a potential new target and suggest that Mtr plays a role during MABC infection.

Assessment of the implementation of safe medication practices in Intensive Medicine Units.

OBJECTIVE: To assess the level of implementation of medication safety practices in Intensive Care Units (ICUs) and to identify opportunities for improvement. DESIGN: A descriptive multicenter study was carried out. SETTING: Intensive Care Units. PARTICIPANTS/PROCEDURE: A total of 40 ICUs voluntarily completed the "Medication use-system safety self-assessment for Intensive Care Units" between March and September 2020. The survey comprised 147 items for evaluation grouped into 10 key elements. MAIN VARIABLES: Calculation was made of the mean scores and mean percentages based on the maximum possible values for the overall survey, referred to the key elements and to each individual item for evaluation. RESULTS: The mean score of the overall questionnaire among the participating ICUs was 436.8 (49.2% of the maximum possible score). No differences were found according to functional dependence, size of the hospital or type of ICU. The key elements referred to the incorporation of clinical pharmacists in these Units, as well as the competence and training of the professionals in safety practices yielded the lowest values (31.2% and 33.2%, respectively). Three other key elements related to accessibility to information about patients and medicines; to the standardization, storage and distribution of medicines; and to the quality and risk management programs, yielded percentages <50%. CONCLUSIONS: Numerous effective safety medication practices have been identified with a low level of implementation in ICUs. This situation must be addressed in order to reduce medication errors in critically ill patients.

Assessment of the implementation of safe medication practices in Intensive Medicine Units. / Evaluación de la implantación de prácticas seguras con los medicamentos en los Servicios de Medicina Intensiva.

OBJECTIVE: To assess the level of implementation of medication safety practices in Intensive Care Units (ICUs) and to identify opportunities for improvement. DESIGN: A descriptive multicenter study was carried out. SETTING: Intensive Care Units. PARTICIPANTS/PROCEDURE: A total of 40 ICUs voluntarily completed the "Medication use-system safety self-assessment for Intensive Care Units" between March and September 2020. The survey comprised 147 items for evaluation grouped into 10 key elements. MAIN VARIABLES: Calculation was made of the mean scores and mean percentages based on the maximum possible values for the overall survey, for the key elements and for each individual item for evaluation. RESULTS: The mean score of the overall questionnaire among the participating ICUs was 436.8 (49.2% of the maximum possible score). No differences were found according to functional dependence, size of the hospital or type of ICU. The key elements referred to the incorporation of clinical pharmacists in these units, as well as the competence and training of the professionals in safety practices yielded the lowest values (31.2% and 33.2%, respectively). Three other key elements related to accessibility to information about patients and medicines; to the standardization, storage and distribution of medicines; and to the quality and risk management programs, yielded percentages below 50%. CONCLUSIONS: Numerous effective safety medication practices have been identified with a low level of implementation in ICUs. This situation must be addressed in order to reduce medication errors in critically ill patients.

The search for novel treatment strategies for Streptococcus pneumoniae infections.

This review provides an overview of the most important novel treatment strategies against Streptococcus pneumoniae infections published over the past 10 years. The pneumococcus causes the majority of community-acquired bacterial pneumonia cases, and it is one of the prime pathogens in bacterial meningitis. Over the last 10 years, extensive research has been conducted to prevent severe pneumococcal infections, with a major focus on (i) boosting the host immune system and (ii) discovering novel antibacterials. Boosting the immune system can be done in two ways, either by actively modulating host immunity, mostly through administration of selective antibodies, or by interfering with pneumococcal virulence factors, thereby supporting the host immune system to effectively overcome an infection. While several of such experimental therapies are promising, few have evolved to clinical trials. The discovery of novel antibacterials is hampered by the high research and development costs versus the relatively low revenues for the pharmaceutical industry. Nevertheless, novel enzymatic assays and target-based drug design, allow the identification of targets and the development of novel molecules to effectively treat this life-threatening pathogen.

Immunosuppression of Syrian golden hamsters accelerates relapse but not the emergence of resistance in Leishmania infantum following recurrent miltefosine pressure.

Although miltefosine (MIL) has only been approved for the treatment of visceral leishmaniasis (VL) in 2002, its application in monotherapy already led to the development of two confirmed MIL-resistant isolates by 2009. Although liposomal amphotericin B is recommended as first-line treatment in Europe, MIL is still occasionally used in HIV co-infected patients. Since their immune system is incapable of controlling the infection, high parasite burdens and post-treatment relapses are common. Linked to the particular pharmacokinetic profile of MIL, successive treatment of recurrent relapses could in principle facilitate the emergence of drug resistance. This study evaluated the effect of immunosuppression (cyclophosphamide 150 mg/kg once weekly) on the development of MIL-resistance in Syrian golden hamsters infected with Leishmania infantum. The hamsters were treated with MIL (20 mg/kg orally for 5 days) whenever clinical signs of infection or relapse were observed. The immunosuppression resulted in a significant depletion of CD4+ lymphocytes and MHCII-expressing cells in peripheral blood, and a concomitant increase in tissue parasite burdens and shorter time to relapse, but the strain's susceptibility upon repeated MIL exposure remained unaltered. This study demonstrates that immunosuppression accelerates the occurrence of relapse without expediting MIL resistance development.

Streptococcus pneumoniae galU gene mutation has a direct effect on biofilm growth, adherence and phagocytosis in vitro and pathogenicity in vivo.

Streptococcus pneumoniae, the most common cause of bacterial pneumonia, has developed a wide range of virulence factors to evade the immune system of which the polysaccharide capsule is the most important one. Formation of this capsule is dependent on the cps gene locus, but also involves other genes-like galU. The pyrophosphorylase encoded by galU plays a role in the UDP-glucose metabolism of prokaryotes and is required for the biosynthesis of capsular polysaccharides. In this paper, the effect of a galU mutation leading to a dysfunctional UDP-glucose pyrophosphorylase (UDPG:PP) on in vitro biofilm biomass, adherence to lung epithelial cells and macrophage phagocytosis is studied. Last, in vivo virulence using a Galleria mellonella model has been studied. We show that the mutation improves streptococcal adherence to epithelial cells and macrophage phagocytosis in vitro, while there is no definitive correlation on biofilm formation between parent and mutant strains. Moreover, in vivo virulence is attenuated for all mutated strains. Together, these results demonstrate that a galU mutation in S. pneumoniae influences host cell interactions in vitro and in vivo and can strongly influence the outcome of a streptococcal infection. As such, UDPG:PP is worth investigating further as a potential drug target.

Impact of primary mouse macrophage cell types on Leishmania infection and in vitro drug susceptibility.

Primary mouse macrophages are frequently used to provide an in vitro intracellular model to evaluate antileishmanial drug efficacy. The present study compared the phenotypic characteristics of Swiss, BALB/c, and C57BL/6 mouse bone marrow-derived macrophages and peritoneal exudate cells using different stimulation and adherence protocols upon infection with a Leishmania infantum laboratory strain and two clinical isolates. Evaluation parameters were susceptibility to infection, permissiveness to amastigote multiplication, and impact on drug efficacy. Observed variations in infection of peritoneal exudate cells can mostly be linked to changes in the inflammatory cytokine profiles (IL-6, TNF-α, KC/GRO) rather than to differences in initial production of nitric oxide and reactive oxygen species. Optimization of the cell stimulation and adherence conditions resulted in comparable infection indices among peritoneal exudate cells and the various types of bone marrow-derived macrophages. BALB/c-derived bone marrow-derived macrophages were slightly more permissive to intracellular amastigote replication. Evaluation of antileishmanial drug potency in the various cell systems revealed minimal variation for antimonials and paromomycin, and no differences for miltefosine and amphotericin B. The study results allow to conclude that drug evaluation can be performed in all tested primary macrophages as only marginal differences are observed in terms of susceptibility to infection and impact of drug exposure. Combined with some practical considerations, the use of 24-h starch-stimulated, 48-h adhered, Swiss-derived peritoneal exudate cells can be advocated as an efficient, reliable, relatively quick, and cost-effective tool for routine drug susceptibility testing in vitro whenever the use of primary cells is feasible.

Handover in Intensive Care. / Traspaso de información en Medicina Intensiva.

Handover is a frequent and complex task that also implies the transfer of the responsibility of the care. The deficiencies in this process are associated with important gaps in clinical safety and also in patient and professional dissatisfaction, as well as increasing health cost. Efforts to standardize this process have increased in recent years, appearing numerous mnemonic tools. Despite this, local are heterogeneous and the level of training in this area is low. The purpose of this review is to highlight the importance of IT while providing a methodological structure that favors effective IT in ICU, reducing the risk associated with this process. Specifically, this document refers to the handover that is established during shift changes or nursing shifts, during the transfer of patients to other diagnostic and therapeutic areas, and to discharge from the ICU. Emergency situations and the potential participation of patients and relatives are also considered. Formulas for measuring quality are finally proposed and potential improvements are mentioned especially in the field of training.

Combining experimental and modelling approaches to study the sources of reactive species induced in water by the COST RF plasma jet.

The vast biomedical potential of cold atmospheric pressure plasmas (CAPs) is governed by the formation of reactive species. These biologically active species are formed upon the interaction of CAPs with the surroundings. In biological milieu, water plays an essential role. The development of biomedical CAPs thus requires understanding of the sources of the reactive species in aqueous media exposed to the plasma. This is especially important in case of the COST RF plasma jet, which is developed as a reference microplasma system. In this work, we investigated the formation of the OH radicals, H atoms and H2O2 in aqueous solutions exposed to the COST plasma jet. This was done by combining experimental and modelling approaches. The liquid phase species were analysed using UV-Vis spectroscopy and spin trapping with hydrogen isotopes and electron paramagnetic resonance (EPR) spectroscopy. The discrimination between the species formed from the liquid phase and the gas phase molecules was performed by EPR and 1H-NMR analyses of the liquid samples. The concentrations of the reactive species in the gas phase plasma were obtained using a zero-dimensional (0D) chemical kinetics computational model. A three-dimensional (3D) fluid dynamics model was developed to provide information on the induced humidity in the plasma effluent. The comparison of the experimentally obtained trends for the formation of the species as a function of the feed gas and effluent humidity with the modelling results suggest that all reactive species detected in our system are mostly formed in the gas phase plasma inside the COST jet, with minor amounts arising from the plasma effluent humidity.

Physician staffing needs in critical care departments. / Estimación de las necesidades de profesionales médicos en los servicios de medicina intensiva.

Departments of Critical Care Medicine are characterized by high medical assistance costs and great complexity. Published recommendations on determining the needs of medical staff in the DCCM are based on low levels of evidence and attribute excessive significance to the structural/welfare approach (physician-to-beds ratio), thus generating incomplete and minimalistic information. The Spanish Society of Intensive Care Medicine and Coronary Units established a Technical Committee of experts, the purpose of which was to draft recommendations regarding requirements for medical professionals in the ICU. The Technical Committee defined the following categories: 1) Patient care-related aspects; 2) Activities outside the ICU; 3) Patient safety and clinical management aspects; 4) Teaching; and 5) Research. A subcommittee was established with experts pertaining to each activity category, defining criteria for quantifying the percentage time of the intensivists dedicated to each task, and taking into account occupational category. A quantitative method was applied, the parameters of which were the number of procedures or tasks and the respective estimated indicative times for patient care-related activities within or outside the context of the DCCM, as well as for teaching and research activities. Regarding non-instrumental activities, which are more difficult to evaluate in real time, a matrix of range versus productivity was applied, defining approximate percentages according to occupational category. All activities and indicative times were tabulated, and a spreadsheet was created that modified a previously designed model in order to perform calculations according to the total sum of hours worked and the hours stipulated in the respective work contract. The competencies needed and the tasks which a Department of Critical Care Medicine professional must perform far exceed those of a purely patient care-related character, and cannot be quantified using structural criteria. The method for describing the 5 types of activity, the quantification of specific tasks, the respective times needed for each task, and the generation of a spreadsheet led to the creation of a management instrument.

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells.

In vitro antiprotozoal activity and cytotoxicity of extracts and isolated constituents from Greenwayodendron suaveolens.

ETHNOPHARMACOLOGICAL RELEVANCE: The Nkundo people (Nkundo area of Bolongo, Mai-Ndombe district, Bandundu Province, DR Congo) use various plant parts of the tree Greenwayodendron suaveolens (Engl. & Diels) Verdc. (syn. Polyalthia suaveolens Engl. & Diels) (Annonaceae) against malaria, but its antiprotozoal constituents are not known. MATERIALS AND METHODS: The crude 80% ethanol extract from the fruits, leaves, root bark and stem bark and 16 fractions were assessed in vitro for their antiprotozoal activity against Trypanosoma brucei brucei, T. cruzi, Leishmania infantum and the chloroquine and pyrimethamine-resistant K1 strain of Plasmodium falciparum (Pf-K1). Their cytotoxic effects were evaluated against MRC-5 cells. Active constituents were isolated by chromatographic means, identified using spectroscopic methods, and evaluated in the same assays. RESULTS: The root bark extract showed the highest activity against P. falciparum K1 (IC50 0.26µg/mL) along with the stem bark alkaloid fraction (IC50 0.27µg/mL). The root bark alkaloid fraction had a pronounced activity against all selected protozoa with IC50 values <1µg/mL. The 90% methanol fractions of the different plant parts showed a pronounced activity against P. falciparum K1, with IC50 values ranging between 0.36µg/mL and 0.69µg/mL. Four constituents were isolated: the triterpenes polycarpol, and dihydropolycarpol, the latter one being reported for the first time from nature, and the alkaloids polyalthenol and N-acetyl-polyveoline. They were active to a various degree against one or more protozoa, mostly accompanied by cytotoxicity. The highest selectivity was observed for N-acetyl-polyveoline against P. falciparum K1 (IC50 2.8µM, selectivity index 10.9). CONCLUSIONS: These results may explain at least in part the traditional use of this plant species against parasitic diseases such as malaria in DR Congo.

Dysregulation of the renin-angiotensin system during lung ischemia-reperfusion injury.

UNLABELLED: Aim/Purpose of the Study: Activation of the renin-angiotensin system leading to increased angiotensin-(1-7) (Ang-(1-7)) and decreased angiotensin 2 (Ang 2) levels may be a new therapeutic approach to reduce acute lung injury. Prolylcarboxypeptidase (PRCP) and prolyloligopeptidase (PREP) are capable of hydrolyzing Ang 2 into Ang-(1-7). However, their relation with circulating Ang 2 levels after lung ischemia-reperfusion injury (LIRI) has never been explored. This study determines whether the activity and expression of PRCP and PREP in plasma and lung tissue is related to circulating Ang 2 levels in a murine model of LIRI. MATERIALS AND METHODS: LIRI in Swiss mice (6 animals per group) was induced by temporary left lung hilar clamping (1 h) followed by 0, 1 or 24 h of reperfusion. Animals in the sham group received thoracotomy only. PRCP activity was measured via RP-HPLC, PREP activity using a fluorogenic substrate and plasma Ang 2 levels via ELISA. Western blotting was used to determine the PRCP and PREP protein expression profiles in left lung tissue. RESULTS: Plasma Ang 2 levels significantly rise after lung ischemia and remain increased after 1 h and 24 h of reperfusion compared to the sham group. While a significant decrease in plasma PREP activity was found after 24 h of reperfusion, a transient increase in plasma PRCP activity was observed after ischemia. However, no correlation with plasma Ang 2 levels could be demonstrated. The activity profiles of PRCP and PREP and the protein expression of PRCP in the lung tissues remained unchanged after LIRI. CONCLUSIONS: LIRI causes a dysregulation of circulating Ang 2 levels and plasma PREP activity, although no direct link between both phenomena could be shown. The activity profile of pulmonary PRCP and PREP was not significantly changed after LIRI, which implies a minor role for local PRCP and PREP in the ischemic lung itself.

Evidence of a drug-specific impact of experimentally selected paromomycin and miltefosine resistance on parasite fitness in Leishmania infantum.

OBJECTIVES: Although miltefosine and paromomycin were only recently introduced to treat visceral leishmaniasis, increasing numbers of miltefosine treatment failures and occasional primary resistance to both drugs have been reported. Understanding alterations in parasite behaviour linked to drug resistance is essential to assess the propensity for emergence and spread of resistant strains, particularly since a positive effect on fitness has been reported for antimony-resistant parasites. This laboratory study compared the fitness of a drug-susceptible parent WT clinical Leishmania infantum isolate (MHOM/FR/96/LEM3323) and derived miltefosine and paromomycin drug-resistant lines that were experimentally selected at the intracellular amastigote level. METHODS: Parasite fitness of WT, paromomycin-resistant and miltefosine-resistant strains, in vitro and in vivo parasite growth, metacyclogenesis, infectivity and macrophage stress responses were comparatively evaluated. RESULTS: No significant differences in promastigote fitness were noted between the WT and paromomycin-resistant strain, while clear benefits could be demonstrated for paromomycin-resistant amastigotes in terms of enhanced in vitro and in vivo growth potential and intracellular stress response. The miltefosine-resistant phenotype showed incomplete promastigote metacyclogenesis, decreased intracellular growth and weakened stress response, revealing a reduced fitness compared with WT parent parasites. CONCLUSIONS: The rapid selection and fitness advantages of paromomycin-resistant amastigotes endorse the current use of paromomycin in combination therapy. Although a reduced fitness of miltefosine-resistant strains may explain the difficulty of miltefosine resistance selection in vitro, the growing number of miltefosine treatment failures in the field still requires further exploratory research.

Synthesis and anti-tubercular activity of N(2)-arylbenzo[g]isoquinoline-5,10-dione-3-iminium bromides.

Tuberculosis has remained a challenge for medicinal chemists worldwide. In the framework of a collaborative program to identify and evaluate novel antitubercular candidate compounds, the biological properties of benzo[g]isoquinoline-5,10-diones have been found to be very promising. In this paper we have further expanded the library by incorporation of an amidinium moiety into the benzo[g]isoquinoline-5,10-dione scaffold. The presence of this functional group also increased the solubility of the quinones in polar solvents. To this purpose N(2)-arylbenzo[g]isoquinoline-5,10-dione-3-iminium bromides were synthesized in a straightforward way by means of a reaction of anilines with 2-(bromomethyl)-3-(cyanomethyl)-1,4-dimethoxynaphthalene. Following the biological evaluation, N(2)-(4-chlorophenyl)-5,10-dioxobenzo[g]isoquinoline-3(2H)-iminium bromide (MIC = 1.16 µM, CC50 = 28.51 µM, SI = 24.58) was selected as the most promising representative. Apart from the nano-molar anti-mycobacterial activity, the compound was able to target intracellular residing Mycobacterium tuberculosis and the susceptibility of a multi-drug-resistant strain towards the compound was confirmed.

In vitro antiprotozoal activity and cytotoxicity of extracts and fractions from the leaves, root bark and stem bark of Isolona hexaloba.

ETHNOPHARMACOLOGICAL RELEVANCE: Isolona hexaloba (Pierre) Engl. and Diels (Annonaceae) is traditionally used in D.R. Congo against parasitic diseases including malaria. MATERIALS AND METHODS: Two crude aqueous extracts, 3 crude methanol extracts and 3 crude 80% ethanol extracts from the leaves, root bark and stem bark together with 12 subfractions from the crude 80% ethanol extracts were evaluated in vitro for their antiprotozoal activity against Trypanosoma brucei brucei, T. cruzi, Leishmania infantum and the chloroquine and pyrimethamine resistant K1 strain of Plasmodium falciparum. Their cytotoxic effects against MRC-5 cell lines were also assessed. RESULTS: Results indicated that the most pronounced activities against T. b. brucei were recorded for the crude methanol extracts of root bark (IC50=1.97 µg/ml; SI>32.49) and leaves (IC50=2.65 µg/ml; SI>24.15). Three samples displayed good activity against T. cruzi: the 80% methanol extract of leaves (IC50=8.33 µg/ml; SI>3.92), its petroleum ether fraction (IC50=8.50 µg/ml; SI=2.52) and the crude aqueous extract of the stem bark (IC50=9.31 µg/ml; SI=3.46). The crude aqueous extract of the leaves exhibited a pronounced and selective activity against L. infantum (IC50=2.00 µg/ml; SI>32). The crude methanol extract of leaves (IC50=6.35 µg/ml; SI>10.10) and the 2 dichloromethane soluble fractions of the 80% ethanol extracts from root bark (IC50=6.96 µg/ml; SI=6.1) and stem bark (IC50=8 µg/ml; SI>8.00) showed good activity and selectivity against L. infantum. The most active samples against Plasmodium falciparum K1 were the leaves crude 80% ethanol extract (0.92 µg/ml) and its fractions: alkaline aqueous (IC50=0.27 µg/ml), 90% methanol (0.90 µg/ml) and dichloromethane (1.04 µg/ml), respectively, with promising selectivity indexes of 35

Lack of correlation between the promastigote back-transformation assay and miltefosine treatment outcome.

OBJECTIVES: Widespread antimony resistance in the Indian subcontinent has enforced a therapy shift in visceral leishmaniasis treatment primarily towards miltefosine and secondarily also towards paromomycin. In vitro selection of miltefosine resistance in Leishmania donovani turned out to be quite challenging. Although no increase in IC50 was detected in the standard intracellular amastigote susceptibility assay, promastigote back-transformation remained positive at high miltefosine concentrations, suggesting a more 'resistant' phenotype. This observation was explored in a large set of Nepalese clinical isolates from miltefosine cure and relapse patients to assess its predictive value for patient treatment outcome. METHODS: The predictive value of the promastigote back-transformation for treatment outcome of a set of Nepalese L. donovani field isolates (n = 17) derived from miltefosine cure and relapse patients was compared with the standard susceptibility assays on promastigotes and intracellular amastigotes. RESULTS: In-depth phenotypic analysis of the clinical isolates revealed no correlation between the different susceptibility assays, nor any clear link to the actual treatment outcome. In addition, the clinical isolates proved to be phenotypically heterogeneous, as reflected by the large variation in drug susceptibility among the established clones. CONCLUSIONS: This in vitro laboratory study shows that miltefosine treatment outcome is not necessarily exclusively linked with the susceptibility profile of pre-treatment isolates, as determined in standard susceptibility assays. The true nature of miltefosine treatment failures still remains ill defined.

In Vivo Selection of Paromomycin and Miltefosine Resistance in Leishmania donovani and L. infantum in a Syrian Hamster Model.

In 2002 and 2006, respectively, miltefosine (MIL) and paromomycin (PMM) were licensed in the Indian subcontinent for treatment of visceral leishmaniasis; however, their future routine use might become jeopardized by the development of drug resistance. Although experimental selection of resistant strains in vitro has repeatedly been reported using the less relevant promastigote vector stage, the outcome of resistance selection on intracellular amastigotes was reported to be protocol and species dependent. To corroborate these in vitro findings, selection of resistance in Leishmania donovani and Leishmania infantum was achieved by successive treatment/relapse cycles in infected Syrian golden hamsters. For PMM, resistant amastigotes were already obtained within 3 treatment/relapse cycles, while their promastigotes retained full susceptibility, thereby sharing the same phenotypic characteristics as in vitro-generated PMM-resistant strains. For MIL, even five treatment/relapse cycles failed to induce significant susceptibility changes in either species, which also corresponds with the in vitro observations where selection of an MIL-resistant phenotype proved to be quite challenging. In conclusion, these results argue for cautious use of PMM in the field to avoid rapid emergence of primary resistance and highlight the need for additional research on the mechanisms and dynamics of MIL resistance selection.

Intracellular amastigote replication may not be required for successful in vitro selection of miltefosine resistance in Leishmania infantum.

Although miltefosine (MIL) has only recently been positioned as a first-line therapeutic option for visceral leishmaniasis, field reports note an increasing trend in treatment failures. Study of laboratory selected MIL-resistant strains is needed in the absence of confirmed resistant clinical isolates. In contrast to promastigotes, experimental in vitro selection of MIL-resistance on intracellular amastigotes has not yet been documented. This study reports for the first time the selection of MIL-resistance in Leishmania infantum LEM3323, a strain which clearly shows active intracellular replication. Starting from the hypothesis that active multiplication may be essential in the resistance selection process; several other L. infantum strains were evaluated. Although strain LEM5269 showed only marginally lower intracellular multiplication, selection for resistance failed, as was also the case for several other strains showing poor or no intracellular replication. These results suggest that intracellular multiplication may not be an absolute prerequisite for the outcome of experimental in vitro MIL-resistance selection in clinical field isolates.

Analysis of contributing factors associated to related patients safety incidents in Intensive Care Medicine.

OBJECTIVE: To explore contributing factors (CF) associated to related critical patients safety incidents. DESIGN: SYREC study pos hoc analysis. SETTING: A total of 79 Intensive Care Departments were involved. PATIENTS: The study sample consisted of 1.017 patients; 591 were affected by one or more incidents. MAIN VARIABLES: The CF were categorized according to a proposed model by the National Patient Safety Agency from United Kingdom that was modified. Type, class and severity of the incidents was analyzed. RESULTS: A total 2,965 CF were reported (1,729 were associated to near miss and 1,236 to adverse events). The CF group more frequently reported were related patients factors. Individual factors were reported more frequently in near miss and task related CF in adverse events. CF were reported in all classes of incidents. The majority of CF were reported in the incidents classified such as less serious, even thought CF patients factors were associated to serious incidents. Individual factors were considered like avoidable and patients factors as unavoidable. CONCLUSIONS: The CF group more frequently reported were patient factors and was associated to more severe and unavoidable incidents. By contrast, individual factors were associated to less severe and avoidable incidents. In general, CF most frequently reported were associated to near miss.