The Innovative Medicines Initiative's New Drugs for Bad Bugs programme: European public-private partnerships for the development of new strategies to tackle antibiotic resistance.

Antibiotic resistance (ABR) is a global public health threat. Despite the emergence of highly resistant organisms and the huge medical need for new drugs, the development of antibacterials has slowed to an unacceptable level worldwide. Numerous government and non-government agencies have called for public-private partnerships and innovative funding mechanisms to address this problem. To respond to this public health crisis, the Innovative Medicines Initiative Joint Undertaking programme has invested more than €660 million, with a goal of matched contributions from the European Commission and the European Federation of Pharmaceutical Industries and Associations, in the development of new antibacterial strategies. The New Drugs for Bad Bugs (ND4BB) programme, an Innovative Medicines Initiative, has the ultimate goal to boost the fight against ABR at every level from basic science and drug discovery, through clinical development to new business models and responsible use of antibiotics. Seven projects have been launched within the ND4BB programme to achieve this goal. Four of them will include clinical trials of new anti-infective compounds, as well as epidemiological studies on an unprecedented scale, which will increase our knowledge of ABR and specific pathogens, and improve the designs of the clinical trials with new investigational drugs. The need for rapid concerted action has driven the funding of seven topics, each of which should add significantly to progress in the fight against ABR. ND4BB unites expertise and provides a platform where the commitment and resources required by all parties are streamlined into a joint public-private partnership initiative of unprecedented scale.

Thymus vulgaris essential oil and thymol inhibit biofilms and interact synergistically with antifungal drugs against drug resistant strains of Candida albicans and Candida tropicalis.

Role of biofilm in disease development and enhance tolerance to antifungal drugs among Candida species has necessitated search for new anti-fungal treatment strategy. Interference in pathogenic biofilm development by new antifungal compounds is considered as an attractive anti-infective strategy. Therefore, the objective of this study was to evaluate Thymus vulgaris essential oil and its major active compound, thymol for their potential to inhibit and eradicate biofilms alone and in combination with antifungal drugs against Candida spp. with especial reference to Candida tropicalis. Anti-candidal efficacy of T. vulgaris and thymol in terms of minimum inhibitory concentration (MIC) was first determined to select the sub-MICs against C. albicans and C. tropicalis. Biofilm formation in the presence and absence of test agents was determined in 96-well microtiter plate by XTT reduction assay and effect of essential oils at sub-MICs of the test agents on biofilm development on glass surface was analysed by light and scanning electron microscopy. Synergistic interaction between essential oils and antifungal drugs were studied by checkerboard method. Effect of sub-MIC of T. vulgaris (0.5×MIC) and thymol (0.5×MIC) on biofilm formation showed a significant reduction (P<0.05) in biofilms. Light microscopy and SEM studies revealed disaggregation and deformed shape of C. albicans biofilm cells and reduced hyphae formation in C. tropicalis biofilm cells at sub-MICs of thymol. Significant effect of T. vulgaris and thymol was also recorded on pre-formed biofilms of both C. albicans and C. tropicalis. T. vulgaris and thymol also showed synergy with fluconazole against both in planktonic and biofilm mode of growth of C. albicans and C. tropicalis. However, synergy with amphotericin B is clearly evident only in planktonic Candida cells. Thyme oil and thymol alone or in combination with antifungal drugs can act as promising antibiofilm agent against drug resistant strains of Candida species and needs further in vivo study to synergise its therapeutic efficacy.

Age, body weight and ovarian function affect oocyte size and morphology in non-PCOS patients undergoing intracytoplasmic sperm injection (ICSI).

The size of oocytes was previously reported to be smaller in obese women with polycystic ovary syndrome (PCOS). In the present prospective cohort study, we sought to determine whether oocyte size and morphology are associated with patient characteristics in non-PCOS women. Oocyte and oolemmal diameter were measured, enlarged perivitelline space (PVS) and ooplasmic granulation were assessed in 308 MII oocytes from 77 IVF/ICSI couples. Statistical analysis was undertaken using SAS version 9.4 (SAS institute Inc., USA). Continuous values are presented as mean ± SD and compared using a two-sample t-test or Mann-Whitney U test as appropriate. Categorical parameters are presented as proportions and compared using a Fisher exact test. Logistic and linear regression models were used to control for the effect of age for categorical and continuous variables respectively. P-value < 0.05 was considered statistically significant. Patients presented with a mean age of 40.3±5.0 years, had a BMI of 25.1±6.1 kg/m2, median AMH levels of 0.6 ng/ml and produced a median of 4 oocytes. Mean total oocyte diameter was 163.2±7.4 µm (range 145.8-182.1 µm), while oolemmal diameter was 109.4±4.1 µm (range 98.5-122.3 µm). After adjusting for age and ovarian reserve increasing BMI was associated with decreased total oocyte diameter (p<0.05). Total oocyte diameter was also inversely associated with AMH levels (p = 0.03) and oocyte yield (p = 0.04). In contrast to total oocyte diameter, oolemmal diameter was not related to patient characteristics. Younger women and those with large oocyte yields demonstrated fewer oocytes with ooplasmic granulation (p<0.05 and p = 0.01). After adjustments for age, ooplasmic granulation was also less frequently observed in oocytes from women with higher AMH (p = 0.03) and increasing BMI (p<0.01). Fertilization was more likely in oocytes with larger oolemmal diameter (p = 0.008). Embryos from oocytes with larger total and ooplasmic diameters were more likely to be transferred or frozen (p = 0.004 and p = 0.01). In non-PCOS infertile women, BMI and ovarian function relate to total oocyte diameter. These results expand on previously observed associations between oocyte size and BMI in women with PCOS. They indicate the importance of detailed oocyte assessments, which may aid the currently used criteria for embryo selection and help to better understand how oocyte status is associated with later embryo development.

Phase 1 study of MEDI3902, an investigational anti-Pseudomonas aeruginosa PcrV and Psl bispecific human monoclonal antibody, in healthy adults.

OBJECTIVES: MEDI3902 is a bivalent, bispecific human immunoglobulin G1κ monoclonal antibody that binds to both the Pseudomonas aeruginosa PcrV protein involved in host cell cytotoxicity and the Psl exopolysaccharide involved in P. aeruginosa colonization and tissue adherence. MEDI3902 is being developed for the prevention of nosocomial P. aeruginosa pneumonia in high-risk patients. METHODS: This phase 1 dose-escalation study (NCT02255760) evaluated the safety, pharmacokinetics, antidrug antibody (ADA) responses and ex vivo anticytotoxicity and opsonophagocytic killing activities of MEDI3902 after a single intravenous infusion in healthy adults aged 18 to 60 years. Fifty-six subjects were randomized in a 3:1 ratio to receive 250, 750, 1500 or 3000 mg of MEDI3902 or placebo and followed for 60 days afterwards. RESULTS: Treatment-emergent adverse events (TEAEs) were mild or moderate in severity; no serious TEAEs were observed. The most common TEAEs were infusion-related reactions. MEDI3902 exhibited approximately linear pharmacokinetics across the 250, 750 and 1500 mg doses and nonlinear pharmacokinetics between the 1500 and 3000 mg doses. One subject in the 3000 mg group tested positive for ADA on day 61 and had a lower MEDI3902 serum concentration from days 43 to 61 than ADA-negative subjects. Serum anticytotoxicity antibody concentrations and opsonophagocytic killing activity were correlated with MEDI3902 serum concentrations across all doses. CONCLUSIONS: Phase 1 study results of MEDI3902 in healthy subjects support further evaluation of its safety and efficacy in subjects at risk for P. aeruginosa pneumonia.

Herpes Simplex Virus Colitis in a Patient with Newly Diagnosed Crohn's Disease.

Herpesvirus colitis is a known cause of morbidity and mortality amongst immunosuppressed individuals. We present a case of HSV colitis following a diagnosis of Crohn's Disease and methylprednisolone therapy. Diagnosis was confirmed by immunohistochemical staining and supported by polymerase chain reaction (PCR) of cutaneous vesicles. The patient recovered following three weeks of acyclovir.

Hemodynamic Impairment Measured by Positron-Emission Tomography Is Regionally Associated with Decreased Cortical Thickness in Moyamoya Phenomenon.

BACKGROUND AND PURPOSE: Impaired cerebrovascular reactivity has been associated with decreased cortical thickness in patients with arterial occlusive diseases. This study tests the hypothesis that severe hemodynamic impairment, indicated by increased oxygen extraction fraction ratios on positron-emission tomography with 15O tracers, is associated with decreased cortical thickness in patients with Moyamoya phenomenon. MATERIALS AND METHODS: Patients with unilateral or bilateral idiopathic Moyamoya phenomenon were recruited. Oxygen extraction fraction ratio maps were generated from cerebral images of O[15O] counts divided by H2[15O] counts with normalization by corresponding cerebellar counts. The normal range of the oxygen extraction fraction ratio was estimated from historically available healthy control subjects. Cortical thickness was estimated from T1-weighted MR imaging and FreeSurfer. Regional samples of oxygen extraction fraction ratios and cortical thicknesses were drawn using FreeSurfer parcellations, retaining only parcellations from the vascular territory of the middle cerebral artery. RESULTS: Complete MR imaging and PET datasets were available in 35 subjects, including 23 women; the mean age at scanning was 44 years. Patients with Moyamoya phenomenon had a significantly increased regional oxygen extraction fraction ratio compared with 15 healthy control subjects (P < .001). Regional oxygen extraction fraction ratio and age were significant predictors of cortical thickness (P < .001 for each) in a generalized linear mixed-effects model. Using hemisphere averages and patient averages, we found that only age was a significant predictor of cortical thickness (P < .001). CONCLUSIONS: Chronic hemodynamic impairment, as indicated by a higher regional oxygen extraction fraction ratio, was significantly predictive of reduced cortical thickness in mixed-effects analysis of FreeSurfer regions. This phenomenon may be related to reversible metabolic down-regulation.

Autosomal recessive primary microcephaly: an analysis of locus heterogeneity and phenotypic variation.

BACKGROUND AND OBJECTIVES: Locus heterogeneity is well established in autosomal recessive primary microcephaly (MCPH) and to date five loci have been mapped. However, the relative contributions of these loci have not been assessed and genotype-phenotype correlations have not been investigated. DESIGN: A study population of 56 consanguineous families resident in or originating from northern Pakistan was ascertained and assessed by the authors. A panel of microsatellite markers spanning each of the MCPH loci was designed, against which the families were genotyped. RESULTS: The head circumference of the 131 affected subjects ranged from 4 to 14 SD below the mean, but there was little intrafamilial variation among affecteds (+/- 1 SD). MCPH5 was the most prevalent, with 24/56 families consistent with linkage; 2/56 families were compatible with linkage to MCPH1, 10/56 to MCPH2, 2/56 to MCPH3, none to MCPH4, and 18/56 did not segregate with any of the loci. CONCLUSIONS: MCPH5 is the most common locus in this population. On clinical grounds alone, the phenotype of families linked to each MCPH locus could not be distinguished. We have also shown that further MCPH loci await discovery with a number of families as yet unlinked.

Acceptability of prenatal testing and termination of pregnancy in Pakistan.

This study aimed to assess acceptability of prenatal testing (PNT) and termination of pregnancy (TOP) for a range of conditions in Pakistani parents with and without a child with a genetic condition. A structured questionnaire assessing acceptability of PNT and TOP for 30 conditions was completed by 400 Pakistani participants: 200 parents with a child with a genetic condition (100 fathers and 100 mothers) and 200 parents without an affected child (100 fathers and 100 mothers). There was a high level of interest in PNT, where over 80 % of parents in all four study groups would want PNT for the majority of the conditions. There was comparatively less interest in TOP for the same conditions (ranging from 5 to 70 % of parents, with mothers of an affected child being most interested). Parents were most likely to be interested in TOP for conditions at the serious end of the continuum. More than half of the participants in each group would consider TOP for anencephaly and quadriplegia. The interest in PNT and TOP for a range of conditions suggests that rapidly developing PNT technologies are likely to be acceptable in Pakistan, a low-middle income level and Muslim country. The comparatively lower level of interest in TOP for the same conditions highlights ethical dilemmas that such technologies are likely to raise.

The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13.1-13.2.

Primary microcephaly is a clinical diagnosis made when an individual has a head circumference of greater than 3 standard deviations below the age and sex matched population mean, mental retardation but without other associated malformations and no apparent aetiology. The majority of cases of primary microcephaly exhibit an autosomal recessive mode of inheritance. We now demonstrate the genetic heterogeneity of this condition with the identification of a second primary microcephaly locus (MCPH2) on chromosome 19q13.1-13.2 in two multi-affected consanguineous families. The minimum critical region containing the MCPH2 locus is defined by the polymorphic markers D19S416 and D19S420 spanning a region of approximately 7.6 cM.

Fluoroquinolones in paediatrics.

Fluoroquinolones have a broad spectrum of activity against gram-positive, gram-negative, and mycobacterial organisms as well as anaerobes, Mycoplasma, Chlamydia, Ureaplasma, and Legionella spp. They have excellent oral bioavailability, with good tissue penetration, and long elimination half-lives. The experience with fluoroquinolones in paediatrics has been limited because of concerns about arthropathy, based on findings in animal models. However, there has not been a definitive fluoroquinolone-associated case of arthropathy described in the literature. We believe that there are a number of specific paediatric infections in which the clinical efficacy and tolerability of the fluoroquinolones should be further investigated. These include patients with cystic fibrosis who have repeated infections with Pseudomonas spp., patients with pseudomonal and other gram-negative infections such as urinary tract infections and osteomyelitis, and febrile neutropenic patients. Meningeal infections caused by multiple drug-resistant Streptococcus pneumoniae and gram-negative organisms, gastroenteritis due to enteric pathogens, and mycobacterial infections are other potential conditions where fluoroquinolones should be studied in paediatric patients.

Buffy coat PCR for diagnosis of experimental pneumococcal pneumonia.

An immunocompetent murine model of pneumococcal pneumonia and bacteremia was used to evaluate a PCR assay based on amplification of the pneumolysin gene. Mice were treated with trovafloxacin to determine the decline in sensitivity of PCR as lung bacterial concentrations decreased and blood cultures became sterile. Forty-three mice were studied for up to 120 h after start of antibiotic treatment. PCR of buffy coat specimens was more sensitive than PCR of plasma. Only 21% of animals had a positive blood culture, whereas 77% of PCR buffy coat assays were positive. After 48 h of therapy all blood culture specimens were sterile, whereas buffy coat PCR was positive in 57.8% of specimens. PCR of buffy coat specimens was negative in all mice colonized nasally with Streptococcus pneumoniae and in rabbits with Escherichia coli bacteremia. Our results demonstrate that our PCR technique using buffy coat specimens is highly specific for invasive pneumococcal disease and remains positive in the majority of animals for at least 48 h after start of antibiotic therapy.

Elevated cytokine concentrations in the nasopharyngeal and tracheal secretions of children with respiratory syncytial virus disease.

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in infants. The role of inflammatory mediators in the pathogenesis of RSV disease is not well-understood. The present study was designed (1) to determine whether RANTES (regulated on activation, normal T cell expressed and presumably secreted), macrophage-inflammatory protein-1-alpha (MIP-1-alpha), interleukin (IL)-6, IL-8 and IL-10 can be detected in respiratory secretions of children with RSV infection and (2) to assess whether the concentrations of these cytokines in respiratory secretions correlate with white blood cell (WBC) counts and RSV concentrations and with disease severity. METHODS: During the 1996 to 1997 RSV season, we studied prospectively 14 intubated and 14 nonintubated children hospitalized with RSV disease. Nasal wash (NW) and tracheal aspirate (TA) samples were obtained from intubated patients on Hospital Days 1, 3 and 5. NW samples were obtained from nonintubated patients on hospital days 1 and 3. Seven healthy children undergoing elective surgery served as controls. All samples were analyzed for: (1) WBC and differential counts; (2) concentrations of RANTES, MIP-1-alpha, IL-6, IL-8 and IL-10; and (3) quantitative RSV cultures, except in control patients. RESULTS: RANTES, MIP-1-alpha, IL-6, IL-8 and IL-10 were detected in NW and TA samples from all children with RSV infection. The concentrations of these cytokines in samples obtained from children with RSV infection were significantly greater than those in samples obtained from control children. NW WBC counts significantly correlated with NW RANTES, IL-6, IL-8 and IL-10 concentrations, whereas TA WBC counts significantly correlated with TA IL-6, IL-8, IL-10 and MIP-1-alpha concentrations. NW RSV concentrations correlated with NW WBC counts and with NW cytokine concentrations. Among children with RSV infection nonintubated patients had greater NW WBC counts and NW RANTES concentrations than intubated patients. TA RANTES, IL-8 and IL-10 concentrations inversely correlated with clinical markers of RSV disease severity. CONCLUSION: The presence of cytokines in NW and TA samples of children with RSV infection suggests that they have a role in mediating the respiratory tract inflammation induced by RSV. These observations could have implications for designing new therapeutic strategies directed at immunomodulation of RSV disease.

Enzyme-linked immunosorbent assay to assess respiratory syncytial virus concentration and correlate results with inflammatory mediators in tracheal secretions.

OBJECTIVE: We developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of respiratory syncytial virus (RSV) in respiratory secretions in intubated patients infected with RSV. METHODS: We compared the quantitative ELISA and a standardized plaque assay in intubated children <2 years of age who were mechanically ventilated for severe RSV disease and enrolled in a randomized double blind placebo-controlled treatment trial of a monoclonal antibody to the F protein of RSV (palivizumab; Synagis). We also examined the relationship between the concentrations of virus as measured by ELISA and of three inflammatory indices in respiratory secretions (white blood cell count, myeloperoxidase and eosinophilic cationic protein). RESULTS: Quantitative ELISA and plaque assay were highly correlated for both tracheal aspirates (r = 0.67, P = 0.001) and nasal wash specimens (r = 0.75, P = 0.001). Treatment with palivizumab significantly neutralized RSV in tracheal aspirates as measured by plaque assay. In contrast quantitation of RSV by ELISA was not affected by palivizumab treatment. This finding is consistent with results that were obtained in preliminary studies of RSV-containing media treated with monoclonal antibody, where we found that the ELISA measured virus whether antibody-bound or not. The inflammatory indices were not correlated with RSV concentration measured by ELISA or plaque assay. CONCLUSIONS: We conclude that this quantitative ELISA is a potentially useful tool for measurement of RSV concentration in respiratory secretions that may help elucidate the pathophysiology of acute RSV infection. Specific antiviral strategies for the treatment of RSV disease could be evaluated by this method.

Application of the enzyme-linked immunoelectrotransfer blot to filter paper blood spots to estimate seroprevalence of cysticercosis in Bolivia.

An enzyme-linked immunoelectrotransfer blot (EITB) assay was used to study the prevalence of cysticercosis in rural Bolivia. Dried blood spots on filter paper from fingersticks were used as assay samples. Before the serosurvey, experiments were performed to show that samples eluted from dried whole blood on filter paper exhibited no decrease in sensitivity when compared with the more traditional serum samples used in the EITB. Fingerstick blood dried on filter paper is a convenient, economical way of transporting and storing field samples for epidemiologic surveys of cysticercosis in developing countries. This report shows the utility of this sample collection method in underdeveloped countries where refrigeration is not possible and where venipuncture is a problem. Blood was obtained from randomly selected residents in three rural regions of Bolivia: Chuquisaca (n = 1,859), Cochabamba (n = 1,516), and Tarija (n = 1,010). The estimated seroprevalence on 10% of the sample collected for the three regions were 9%, 4.5%, and 2%, respectively.

Progression of phenotype in Leber's congenital amaurosis with a mutation at the LCA5 locus.

BACKGROUND: Leber's congenital amaurosis (LCA) accounts for 5% of inherited retinal disease and is usually inherited as an autosomal recessive trait. Genetic and clinical heterogeneity exist. Mutations have been described in the RPE65, CRB1, RPGRIP1, AIPL1, GUCY2D, and CRX genes and other pedigrees show linkage to the LCA3 and LCA5 loci. The latter is a new locus which maps to 6q11-q16. The ocular findings and the evolution of the macula staphyloma are described in five members of a Pakistani family with consanguinity and a mutation in the LCA5 gene. METHODS: 13 family members including five affected individuals consented to DNA analysis and ocular examination including fundal photography. RESULTS: Ocular abnormalities are described. The most striking feature was the progression of macula abnormalities in three brothers resulting in a colobomatous appearance in the eldest compared to only mild atrophy in the youngest. The phenotypic pattern of this mutation in this Pakistani family contrasts with the "Old Order River Brethren" who were of Swiss descent, in whom the mutation was first described. CONCLUSION: The evolution of a new phenotypic picture is presented to a mutation in LCA5.

[Asthma and respiratory syncytial virus. New opportunities for therapeutic intervention]. / Asma y virus respiratorio sincitial. Nuevas oportunidades de intervención terapéutica.

Numerous studies have described an association between respiratory sincticial virus (RSV) infection in infancy and the subsequent development of airway hyperresponsiveness (AHR). Besides the exaggerated immune response and the abnormal neurogenic mechanisms induced by RSV, recent studies have correlated the "persistence" of RSV in the lower respiratory tract with the development of AHR. Several investigators have evaluated whether treatment with antiviral or immunosuppressive agents could decrease the long term respiratory abnormalities induced by RSV. The RSV murine model has allowed us to study the immunopathogenesis of RSV-induced AHR. Once the airway obstruction, typical of acute disease, is resolved and no virus is longer detected by cell cultures, mice progress into a chronic phase characterized by AHR and persistent airway inflammation. The use of polymerase chain reaction assay for RSV quantitation has demonstrated, quite unexpectedly, the presence of RSV RNA in the lower respiratory tract of mice during the chronic phase of the disease. As an example of intervention, the administration of an anti-RSV neutralizing antibody (palivizumab) was associated with a significant reduction in viral replication, pulmonary inflammation and inflammatory cytokines, as well as a significant improvement in the pulmonary function both in the acute and chronic phases of the disease. Future clinical studies to determine whether therapy with palivizumab can prevent the long-term morbidity associated with RSV in children are warranted.