Possible antiviral effect of ciprofloxacin treatment on polyomavirus BK replication and analysis of non-coding control region sequences.

Acute renal dysfunction (ARD) is a common complication in renal transplant recipients. Multiple factors contribute to ARD development, including acute rejection and microbial infections. Many viral infections after kidney transplantation result from reactivation of "latent" viruses in the host or from the graft, such as the human Polyomavirus BK (BKV). We report the case of a 39 year-old recipient of a 2nd kidney graft who experienced BKV reactivation after a second episode of acute humoral rejection. A 10-day treatment with the quinolone antibiotic ciprofloxacin was administered with an increase of immunosuppressive therapy despite the active BKV replication. Real Time PCR analysis performed after treatment with ciprofloxacin, unexpectedly showed clearance of BK viremia and regression of BK viruria. During the follow-up, BK viremia persisted undetectable while viruria decreased further and disappeared after 3 months.BKV non-coding control region sequence analysis from all positive samples always showed the presence of archetypal sequences, with two single-nucleotide substitutions and one nucleotide deletion that, interestingly, were all representative of the subtype/subgroup I/b-1 we identified by the viral protein 1 sequencing analysis.We report the potential effect of the quinolone antibiotic ciprofloxacin in the decrease of the BKV load in both blood and urine.

Molecular Characterization of Stenotrophomonas maltophilia isolates from cystic fibrosis patients and the hospital environment.

OBJECTIVES: To ascertain whether cystic fibrosis (CF) patients are colonized or infected with unique or multiple strains of Stenotrophomonas maltophilia; to understand whether some strains colonize or infect more than 1 patient, indicating clonal spread; and to explore the molecular heterogeneity of hospital water isolates and their correlation with clinical isolates. SETTING: The regional CF center of Policlinico "Umberto I" of Rome, Italy. METHODS: The study was carried out on a random sample of S. maltophilia isolates (n = 110) collected from CF patients (n = 50) during the period 2002-2005 and on 24 water isolates obtained during a monitoring program in the first 6 months of 2005. Home environmental samplings were not performed. All isolates, which were recovered from cultures of specimens obtained in both inpatient and outpatient settings, were genotyped with DNA macrorestriction analysis with the restriction enzyme XbaI and pulsed-field gel electrophoresis. RESULTS: One-third of the patients with repeated episodes of S. maltophilia infection or colonization hosted more than 1 strain. A potential transmission, defined as the isolation of the same strain in 2 or more patients, occurred 5 times, showing a frequency of potential transmission episodes slightly higher than previously reported. Water, taps, and sinks of the different rooms of the CF center tended to be persistently colonized with the same strain of S. maltophilia, with no correlation between clinical and water-associated isolates. CONCLUSIONS: The study does not provide sufficient data to conclude definitively that isolation of colonized or infected CF patients and control of hospital water systems contamination would be beneficial infection control measures. Epidemiologic analytical studies that correlate the presence of S. maltophilia with clinical outcomes are strongly needed.

Inhibition of Epstein Barr Virus LMP1 gene expression in B lymphocytes by antisense oligonucleotides: uptake and efficacy of lipid-based and receptor-mediated delivery systems.

Epstein Barr Virus (EBV), is associated with an increasing number of lymphoid and epithelial malignancies. Among the genes expressed by EBV during latency, LMP1 plays a key role for growth transformation and immortalization of B lymphocytes. We have previously shown that antisense oligonucleotides (ONs) directed to LMP1 mRNA, effectively suppressed LMP1 gene expression and substantially reduced proliferation of the infected cells. The use of antisense phosphodiester oligonucleotides as therapeutic agents is limited by inefficient cellular uptake and intracellular transport to the target mRNA. We tested the ability of three cationic carriers internalized by different pathways, to increase the delivery of anti-LMP1-ON to their site of action in EBV-infected B lymphocytes. We report here that liposomes, dendrimers or transferrin-polylysine-conjugated ON were internalized by the cells at an extent several fold higher than that of the naked oligomers. However, only the delivery system exploiting the transferrin receptor pathway of internalization, was able to vectorize biologically active antisense LMP1-ON.

Emerging cystic fibrosis pathogens: incidence and antimicrobial resistance.

We examined the frequency of isolation and the antimicrobial resistance of Burkholderia cepacia complex, Stenotrophomonas maltophilia and Achromobacter xylosoxidans in cystic fibrosis patients from 2000 to 2004. Strains susceptibility to tobramycin, piperacillin/tazobactam, imipenem, gentamicin, ciprofloxacin and ceftazidime was determined by disc diffusion assay. B. cepacia complex showed a very high resistance also to ciprofloxacin reaching 100% in 2004. S. maltophilia and A. xvylosoxidans showed high rates of antimicrobial resistance both aminoglycoside and ciprofloxacin. It is very important to monitor the percentage of isolation of these species over time to verify strains resistance to antibiotics and also to test new combinations of antimicrobial agents.

Application of real time PCR in post transplant monitoring of cytomegalovirus infection: comparison with other diagnostic approaches.

Immunosuppressive status in solid organ transplant recipients is often related to the reactivation of Human Cytomegalovirus (HCMV) infection that remains one of the major causes of morbidity and mortality. Therefore, the early detection of HCMV followed by infection monitoring is important to institute prompt and appropriate treatment. In recent years good results have been obtained by HCMV DNA amplification methods; qualitative and quantitative approaches have shown good sensitivity and specificity, but they often require post-PCR manipulation that adds time to the analysis and may lead to contamination problems. Recently, Real Time PCR (RT-PCR) has been proposed in HCMV DNA analysis as a valid method for its good sensitivity and rapidity. In the present study, twenty-five solid organ transplant recipients were analyzed for HCMV diagnosis; 60 peripheral blood leukocytes and 120 plasma samples were tested by RT-PCR and the results compared to those obtained by a qualitative Nested PCR and a quantitative DNA enzyme immunoassay.

Early evidence of lymphoproliferative disorder: post-transplant monitoring of Epstein-Barr infection in adult and pediatric patients.

Transplant patients are at high risk of post-transplant lymphoproliferative disorder (PTLD). A strong correlation between Epstein-Barr virus (EBV) and PTLD is observed in pediatric patients with primary infection after transplant. Because many patients have responded to reversal of immunosuppressive therapy, an early identification of EBV is essential for the reduction of immunosuppression and/or introduction of antiviral therapy to prevent PTLD. Polymerase chain reaction (PCR) is a specific and sensitive method to identify EBV DNA in blood. The aim of our study was to establish a protocol for monitoring EBV infection in transplanted patients for early identification those at high risk of PTLD. Viral presence in peripheral blood leukocytes (PBL) and serum samples was revealed by Nested PCR; positive specimens were quantified with Real Time PCR (RT-PCR). DNA in PBL was observed in 12 cases and 6 showed EBV in sera. Quantitative analysis showed a wide range of EBV DNA copies in leukocytes that were higher than in sera. Two patients displayed high viral load values in both PBL and sera associated with clinical evidence of PTLD. Our data suggest that the study of the EBV load represents an essential approach in the diagnosis of PTLD and the analysis of serum samples could provide useful information in the post-transplant monitoring of high-risk patients.

Protective features of monoclonal antibodies to Escherichia coli during experimental infection of mice with homologous and heterologous serotypes of E. coli.

Murine monoclonal antibodies (MAbs) MT1F and ARM1-4, recognising proteins on the surface of untreated Escherichia coli O6:K-, protected 100% of mice challenged intraperitoneally with 2 x LD50 of the same strain. MAb MT1F protected 70% of animals challenged with 2 x LD50 of E. coli O111:B4, whereas ARM1-4 gave complete protection. Lower survival was observed in mice given either MAb and challenged with E. coli O128:K-, with values ranging from 30 to 42%. However, the protection afforded against E. coli O111:B4 and E. coli O128:K- was significantly improved when the mice were pre-treated with a mixture of the two MAbs. Control mice, pre-treated with unrelated ascitic fluid and challenged with any of the E. coli serotypes, showed 100% mortality and organ histological lesions resembling those of the early stages of septic shock. The mice had high levels of circulating endotoxin and tumour necrosis factor-alpha (TNF-alpha) at 90 min after challenge. In contrast, mice treated with MAbs and surviving the infection displayed moderate histological lesions, enhanced bacterial clearance and lower serum levels of TNF-alpha, despite circulating endotoxin levels that were higher than in the control group. Protection by the MAbs was probably due to the prevention of the bacterial spread to organs and of the cascade of events leading to septic shock. This occurred in spite of the presence of high levels of circulating endotoxin.