Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium

Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.     

Comparison of standard and steerable catheters for bile duct cannulation in ERCP

BACKGROUND AND STUDY AIMS: The aim of the study was to compare two steerable endoscopic retrograde cholangiopancreatography (ERCP) catheters with regard to speed and safety in cannulating the common bile duct. PATIENTS AND METHODS: A standard cannula, a short-nosed sphincterotome, and a bendable catheter were used. At two tertiary centres, a total of 312 patients were randomly assigned to receive treatment with one of three catheters and either by a trainee or an expert endoscopist. When cannulation failed, a further attempt was made with a different catheter. If this failed, a change in operator or other manoeuvres followed. The following were assessed: time to cholangiography and deep cannulation, number of attempts and success rates of cannulation, number of pancreatic duct injections, success of catheter cross-over, and complication rates. RESULTS: Both steerable catheters were significantly better for the initial cholangiogram than the standard catheter (standard catheter 75 %, bendable catheter 84 %, sphincterotome 88 %; P = 0.038), with no significant differences between the bendable catheter and the sphincterotome. Both were also better for deep cannulation of the bile duct (standard cannula 66 %, bendable catheter 69 %, sphincterotome 78 %; P = 0.15). When the standard catheter failed, a steerable catheter succeeded in 26 % of cases. Trainees experienced greater benefit from using steerable catheters. For experts, the bendable catheter was the quickest to achieve cholangiography and deep cannulation. Further manoeuvres had an 85-90 % success rate in allowing biliary access. Twenty of 23 needle-knife papillotomies (87 %) were successful when other methods had failed. The overall ERCP success rate was 97 %. Pancreatitis occurred in 5.3 % of cases. CONCLUSIONS: Steerable catheters allow faster access and can succeed when a standard catheter fails. If cannulation is difficult, changing the catheter should be considered at an early stage. Needle-knife papillotomy is a successful technique in expert hands.     

Efficacy of UK-49,858 (fluconazole) against Candida albicans experimental infections in mice.

UK-49,858 (fluconazole), a new, orally absorbed bis-triazole derivative, has been evaluated against systemic infections with Candida albicans in normal and immunosuppressed mice and against an intestinal infection with C. albicans in immunosuppressed mice. Orally administered ketoconazole was used as a comparison agent throughout, and orally administered amphotericin B was included for comparative in the experimental intestinal infection. In a 10-day dosage regimen, UK-49,858 was far more active than ketoconazole against systemic infections with C. albicans in normal and immunosuppressed mice. In normal mice, extension of UK-49,858 dosing to 30 days resulted in prolongation of survival to over 90 days, and up to 60% of treated animals had no detectable C. albicans in their kidneys. In addition, over 90% of mice with intestinal candidiasis had culture-negative feces after a 3-day treatment with UK-49,858, but only 62 and 23% of mice gave this response after amphotericin B and ketoconazole therapy, respectively. These data suggest that UK-49,858 may be of value in the treatment of systemic and gastrointestinal infections due to C. albicans in humans.     

Extended multilocus variable-number tandem-repeat analysis of Clostridium difficile correlates exactly with ribotyping and enables identification of hospital transmission

PCR ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR ribotyping.     

Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.     

Candidaemia with uncommon Candida species: predisposing factors, outcome, antifungal susceptibility, and implications for management

The risk factors for and clinical features of bloodstream infection with uncommon Candida spp. (species other than C. albicans, C. glabrata, C. parapsilosis, C. tropicals and C. krusei) are incompletely defined. To identify clinical variables associated with these species that might guide management, 57 cases of candidaemia resulting from uncommon Candida spp. were analysed in comparison with 517 episodes of Candida albicans candidaemia (2001–2004). Infection with uncommon Candida spp. (5.3% of candidaemia cases), as compared with C. albicans candidaemia, was significantly more likely to be outpatient-acquired than inpatient-acquired (15 of 57 vs. 65 of 517 episodes, p 0.01). Prior exposure to fluconazole was uncommon (n = 1). Candida dubliniensis was the commonest species (n = 22, 39%), followed by Candida guilliermondii (n = 11, 19%) and Candida lusitaniae (n = 7, 12%). C. dubliniensis candidaemia was independently associated with recent intravenous drug use (p 0.01) and chronic liver disease (p 0.03), and infection with species other than C. dubliniensis was independently associated with age <65 years (p 0.02), male sex (p 0.03) and human immunodeficiency virus infection (p 0.05). Presence of sepsis at diagnosis and crude 30-day mortality rates were similar for C. dubliniensis-related, non-C. dubliniensis-related and C. albicans-related candidaemia. Haematological malignancy was the commonest predisposing factor in C. guilliermondii (n = 3, 27%) and C. lusitaniae (n = 3, 43%) candidaemia. The 30-day mortality rate of C. lusitaniae candidaemia was higher than the overall death rate for all uncommon Candida spp. (42.9% vs. 25%, p not significant). All isolates were susceptible to amphotericin B, voriconazole, posaconazole, and caspofungin; five strains (9%) had fluconazole MIC values of 16–32 mg/L. Candidaemia due to uncommon Candida spp. is emerging among hospital outpatients; certain clinical variables may assist in recognition of this entity.     

Non-Tuberculous Mycobacterial Pulmonary Disease identified during community-based screening for Mycobacterium Tuberculosis: a case report

There is a rising prevalence of Non-Tuberculous Mycobacterial (NTM) disease in sub-Saharan Africa identified on culture specimens. However, distinguishing mycobacterial colonisations from infection from identified NTMs on culture in the sub-Saharan Africa setting remains to be established. A 49-year-old man presented with the cardinal symptoms of tuberculosis (TB) in a community TB prevalence survey in Blantyre, Malawi. Mycobacteriology was atypical, prompting a line probe assay which revealed Mycobacterium avium complex (MAC) species.The epidemiology of Mycobacterium tuberculosis complex (MTBC) is better known than that of NTM. Up-scaling culture and speciation may be a solution to this gap in knowledge of the burden of disease of NTM. Like most resource-poor settings, TB culture is not routinely done in the diagnosis and management of TB in Malawi. Furthermore, the treatment of NTM is not analogous to that of MTBC. The multi-drug regimens used for NTM disease treatment includes a newer macrolide (azithromycin, clarithromycin), ethambutol, and rifamycin, and require prolonged durations of therapy aimed at facilitating clearance of the mycobacteria and minimizing the emergence of drug resistance. Clinicians must thus be aware of this rising burden of NTM disease and consider other diagnostic options to better investigate this disease in patients.     

Brain infusion of lipopolysaccharide increases uterine growth as a function of estrogen replacement regimen: suppression of uterine estrogen receptor-alpha by constant, but not pulsed, estrogen replacement

The effects of estrogen therapy can differ depending on the regimen of estrogen administration. In addition, estrogen can modulate the effects of stressors. To examine the interaction between these systems, we infused adult female rats with lipopolysaccharide (LPS) into the fourth ventricle of the brain for 6 d and compared the effects of constant and pulsed estrogen replacement. Constant, but not pulsed, estrogen treatment reduced estrogen receptor-alpha (ERalpha) protein by 90% in the uterus and increased heat-shock proteins 70 and 90 by 74 and 48%, respectively, whereas progesterone receptor levels increased in all ovariectomized rats receiving estrogen replacement. In contrast to the uterine decline in ERalpha, no changes in ERalpha were observed in the hypothalamus or hippocampus, and ERbeta levels were unchanged in all regions tested. Brain infusion of LPS did not alter these proteins but increased the number of activated microglia in the thalamus and reduced body weight in all rats as well as activated the hypothalamic-pituitary-adrenal axis in ovariectomized rats, as determined by elevations in circulating corticosterone and progesterone. Estrogen treatments did not alter these markers, and no differences were observed in cortical choline acetyltransferase activity or nitrotyrosine for any of the treatment groups. The current study found an unexpected increase in uterine weight in lipopolysaccharide-infused rats treated with constant, but not pulsed, estrogen. This report suggests that constant and pulsed regimens of estrogen administration produce different effects and that stress may be an important factor in the postmenopausal intervention with estrogen.