Detecting latent tuberculosis infection during anti-tumor necrosis factor therapy.

OBJECTIVES: There is little information regarding the reliability of repeat tuberculin skin tests (TSTs) and interferon gamma release assays (IGRAs) in detecting latent tuberculosis infection (LTBI) in people on anti-tumour necrosis factor (TNF) medication. METHODS: We conducted a prospective, observational study of patients referred to the Saskatoon Tuberculosis (TB) Clinic prior to starting anti-TNF medication. A chest x-ray (CXR), 2-step TST and IGRA (QuantiFERON-TB Gold In-Tube Method) were performed at baseline. Those patients with a baseline TST ≥10 mm and/or a positive IGRA were followed with a clinic visit, CXR, TST and IGRA at 3 and 6 months after starting anti-TNF medication. RESULTS: Of 106 potential patients, 91 consented to participate. Twenty-six patients had a positive (≥ 10 mm) TST or IGRA at baseline; twelve started and stayed on anti-TNF medication through the 6-month follow-up and completed both planned follow-up visits. The baseline mean TST measurement for the 12 participants was 13.9 mm (SD 11.4), increasing to a mean of 16.8 mm (SD 9.3) post-booster. At 3 months post-anti-TNF initiation, there was an overall decrease in TST measurement (mean=10.0 mm; SD 9.3; p=0.013), with measurements <5 mm in 3 of the 12 patients. By the 6-month TST, a response recovery was observed with a mean TST measurement of 14.5 mm (SD 7.7), with 11/12 ≥5 mm. The IGRA was unchanged throughout the study period in all patients. The overall agreement between TST and IGRA was poor (kappa coefficient = 0.180, p=0.020). CONCLUSIONS: We demonstrated a transient but significant decrease in TST response in the first six months of anti-TNF therapy.

Methicillin-resistant Staphylococcus aureus replication in the presence of high (≥32 µg/ml) drug concentration of vancomycin as seen by electron microscopy.

Methicillin-resistant Staphylococcus aureus (MRSA) has unfortunately become a common pathogen in many healthcare facilities. In many institutions, vancomycin remains the preferred agent for treating serious MRSA infections including bacteraemia with or without endocarditis. The mutant prevention concentration (MPC) testing ≥109 colony forming units of bacteria, describes the antimicrobial drug concentration blocking the growth of the least susceptible cell from high density bacterial populations. With blood culture isolates of MRSA, we discovered strains with MPC values ≥32 µg/ml and viable cells could be readily recovered from agar plates containing 32 µg/ml of vancomycin. To investigate MRSA strains surviving in high concentrations of vancomycin on drug containing agar plates, we utilized electron microscopy to measure cell wall thickness as this has been previously reported as a potential mechanism of resistance1 along with septum thickening. Our data shows MRSA replication from high density bacterial populations in the presence of ≥32 µg/ml of vancomycin. Such observations may explain vancomycin failure in some patients and/or persistent bacteraemia and could potentially question the use of this drug in some critically ill patients in favour of an alternative agent.

Zebularine inhibits human acute myeloid leukemia cell growth in vitro in association with p15INK4B demethylation and reexpression.

OBJECTIVE: The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine, can reverse p15INK4B promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies. METHODS: We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples. RESULTS: Zebularine treatment of AML193, which has a densely methylated p15INK4B promoter, results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation, arrests cells at G(2)/M, and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis. CONCLUSION: Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies.

5-Aza-2'-deoxycytidine (decitabine) can relieve p21WAF1 repression in human acute myeloid leukemia by a mechanism involving release of histone deacetylase 1 (HDAC1) without requiring p21WAF1 promoter demethylation.

Decitabine is a potent demethylating agent that exhibits clinical activity against myeloid malignancies. Numerous genes silenced by hypermethylation are reactivated by decitabine through a mechanism involving promoter demethylation with subsequent release of histone deacetylases (HDACs) and accumulation of acetylated histones. Recent studies indicating that decitabine also induces regional chromatin remodeling of some unmethylated genes suggest additional mechanisms of action. Decitabine reactivates unmethylated p21WAF1 in some AML cell lines but the possible occurrence of p21WAF1 methylation in AML in vivo has not been studied in detail and decitabine effects on p21WAF1 chromatin remodeling have not been reported. We found that p21WAF1 mRNA was undetectable in 6 of 24 AML patient samples and 4 of 5 AML cell lines but there was no evidence of p21WAF1 promoter methylation. However, decitabine induced p21WAF1 in AML cell lines KG-1 and KG-1a in association with release of HDAC1 and increased acetylated histone H3 at the unmethylated p21WAF1 promoter. Decitabine effects on p21WAF1 histone acetylation and induction were enhanced by the HDAC inhibitor trichostatin A and were independent of wild type p53. Our findings indicate that decitabine can relieve p21WAF1 repression in AML by a mechanism that involves release of HDAC1 without requiring promoter demethylation. Furthermore, our study provides evidence that combined decitabine and HDAC inhibitor treatment can enhance chromatin remodeling and reactivation of an unmethylated tumor suppressor gene. This latter finding is of relevance to the clinical use of these agents in AML as we found the p21WAF1 promoter to be unmethylated in vivo.

Propionibacterium acnes infections after cranial neurosurgery.

BACKGROUND: Propionibacterium acnes (P. acnes) is a relatively avirulent organism that is part of the normal skin flora. Most patient isolates are considered contaminants but, in a small subset of patients, particularly in the post-neurosurgery setting, the organism can cause significant infections. We reviewed our experience with the occurrence and management of P. acnes infections after cranial neurosurgical procedures over a five-year period. METHODS: Patients with positive cultures for P. acnes between 1996 and 2001 were identified by review of the Saskatoon Health Region microbiology laboratory database. Of the 141 positive cultures, a review of hospital records identified six patients with P. acnes infections after neurosurgical procedures. A review of the literature related to P. acnes associated CNS infections was conducted. RESULTS: All patients had undergone a craniotomy or burrhole placement, and one patient had received prior radiotherapy. There were no P. acnes-related ventriculoperitoneal shunt infections. All patients presented with scalp swelling and three had purulent discharge. Symptoms occurred more than two months after the initial surgery in five of six patients, while one patient developed symptoms three years post-operatively. Management for all patients included removal of the craniotomy flap and treatment with parenteral antibiotics, followed in most cases by oral antibiotics. A good response without relapse of infection was seen in five patients; one patient had recurrent infection after cranioplasty. CONCLUSIONS: P. acnes is a rare but important cause of infection after craniotomy. Wound debridement, removal of the bone flap and adequate antibiotic coverage result in cure in the majority of patients.

Quinupristin/dalfopristin.

Gram-positive pathogens are associated with both community- and hospital-acquired infections. These infections may be life-threatening in hospitalised patients, especially in those with significant underlying acute or chronic diseases. Prompt and appropriate antimicrobial therapy is essential for avoiding morbidity and mortality. The concept of appropriate therapy is being redefined by increasing antimicrobial resistance, especially amongst Gram-positive pathogens. This has been most dramatic with penicillin-resistant Streptococcus pneumoniae in the community, including cross-resistance to other classes of antimicrobial agents. In the US, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) with community isolates is significant. For hospital-acquired Gram-positive pathogens, MRSA, vancomycin-resistant Enterococcus species and vancomycin-intermediate resistant and -resistant S. aureus are a great concern, particularly as the frequency of recovery of these pathogens from infected patients increases. The net result of these various resistance issues is a reduction in the number of appropriate antimicrobial agents for treating infected patients. Quinupristin/dalfopristin is a parental streptogramin with a spectrum of activity that includes Gram-positive pathogens, including those resistant to other classes of antimicrobial compounds. In this review, data summarising the frequency of recovered Gram-positive pathogens from various infectious diseases, the escalating prevalence of resistance amongst Gram-positive pathogens and the factors making quinupristin/dalfopristin a suitable agent for treating patients infected with Gram-positive organisms will be discussed.