Vittorio Sambri

Borrelia burgdorferi NapA-driven Th17 cell inflammation in lyme arthritis.

OBJECTIVE Human Lyme arthritis caused by Borrelia burgdorferi is characterized by an inflammatory infiltrate that consists mainly of neutrophils and T cells. This study was undertaken to evaluate the role of the innate and acquired immune responses elicited by the neutrophil-activating protein A (NapA) of B burgdorferi in patients with Lyme arthritis. METHODS Serum anti-NapA antibodies were measured in 27 patients with Lyme arthritis and 30 healthy control subjects. The cytokine profile of synovial fluid T cells specific for NapA was investigated in 5 patients with Lyme arthritis. The cytokine profile induced by NapA in neutrophils and monocytes was also investigated. RESULTS Serum anti-NapA antibodies were found in 48% of the patients with Lyme arthritis but were undetectable in the healthy controls. T cells from the synovial fluid of patients with Lyme arthritis produced interleukin-17 (IL-17) in response to NapA. Moreover, NapA was able to induce the expression of IL-23 in neutrophils and monocytes, as well as the expression of IL-6, IL-1beta, and transforming growth factor beta (TGFbeta) in monocytes, via Toll-like receptor 2. CONCLUSION These findings indicate that NapA of B burgdorferi is able to drive the expression of IL-6, IL-1beta, IL-23, and TGFbeta by cells of the innate immune system and to elicit a synovial fluid Th17 cell response that might play a crucial role in the pathogenesis of Lyme arthritis.

Diagnosis of bloodstream infections in immunocompromised patients by real-time PCR

OBJECTIVES The diagnosis of bloodstream infections (BSIs) in immunocompromised patients, such as patients with cancer, is challenging. Although blood culture (BC) is considered the standard diagnostic tool for BSIs, it takes several days to yield results and has low sensitivity in these patients. Here, we tested a novel method for diagnosing BSIs in a large cohort of immunodepressed patients. METHODS Real-time PCR (LightCycler SeptiFast Test M(GRADE), Roche Diagnostics) was compared with BC for its ability to detect bacteria and fungi in blood samples from 100 immunocompromised patients (98 with cancer) in whom sepsis was suspected. RESULTS In concordant samples (79.2% of total cases), real-time PCR identified the presence or absence of microbes significantly faster than BC (p=3.7x10(-49), t-test). Furthermore, in 6 cases, SeptiFast distinguished contamination of BCs by coagulase-negative staphylococci. SeptiFast, however, failed to detect 5 cases of clinically relevant BSI that tested positive by BC. CONCLUSIONS SeptiFast rapidly diagnosed BSIs in our cohort of immunosuppressed patients. The results of this study suggest that SeptiFast can be used in conjunction with, but cannot replace, BC to better identify the etiology of fever in immunocompromised patients.

A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid

BACKGROUND Usutu virus (USUV), a flavivirus that belongs to the Japanese encephalitis virus (JEV) family, has recently emerged as a human pathogen, necessitating new diagnostic tools. OBJECTIVE The development and assessment of a real-time RT-PCR assay to detect USUV in human samples. STUDY DESIGN Based on USUV genomic sequences from GenBank, USUV-specific primers and probes that target the NS5 gene were designed. The sensitivity was evaluated in a 10-fold dilution series of plasmid that contained the amplicon and in a dilution series of a quantified human USUV isolate. The specificity was determined by testing various concentrations of related ArBo viruses, including flaviviruses and phleboviruses. Human RNAse P was also amplified in the assay. One hundred four human specimens from patients who suffered from viral meningoencephalitis were evaluated. RESULTS The real-time RT-PCR assay had a sensitivity of 50 genomic copies per reaction (corresponding to 2200 copies/ml) and 1 PFU/ml of USUV isolate. USUV isolates from Austria were identified with identical efficiency, and no ArBo viruses, other than USUV, were detected. USUV was also identified in 3 cerebrospinal fluid samples. All human samples were positive for RNAse P. CONCLUSIONS This PCR assay is recommended for all cases in which a rapid and clinically accurate diagnosis of human USUV infection is required.

Laboratory diagnosis of late-onset sepsis in newborns by multiplex real-time PCR.

Bloodstream infections (BSIs) are an important cause of neonatal morbidity and mortality, and often result in prolonged hospitalization of infants who are admitted to neonatal intensive care units (VerboonMaciolek et al., 2006). Late-onset neonatal sepsis (occurring in newborns aged older than 3 days) occurs in approximately 0.1 % of all newborns and in up to ~25 % of very low birth weight infants (birth weight ,1500 g) (Kaufman & Fairchild, 2004). Early diagnosis of sepsis and prompt treatment are critical in preventing severe and life-threatening complications in these patients (Harbarth et al., 2003; Kollef, 2003; Lodise et al., 2003). The clinical recognition of sepsis in neonates is difficult, however, because the signs and symptoms are often non-specific (Gerdes, 1991; Verboon-Maciolek et al., 2006) and blood cultures (BCs) are rarely positive.

Preliminary investigations on a new gentamicin and vancomycin-coated PMMA nail for the treatment of bone and intramedullary infections: An experimental study in the rabbit.

To evaluate a new gentamicin–vancomycin‐ impregnated (2:1) PMMA coating nail as a drug delivery device to treat bone and intramedullary infections, methicillin‐resistant Staphylococcus aureus (MRSA) was used to induce femoral osteomyelitis in 20 New Zealand male rabbits. Four weeks after inoculum, the animals were submitted to debridement of infected femur canal, divided into four groups of five animals each and treated according to the following protocols: Group 1, insertion of a steel AISI316 intramedullary nail; Group 2, insertion of a gentamicin–vancomycin‐impregnated PMMA nail; Group 3, no therapy; and Group 4 no fixation device and 1‐week systemic antibiotic therapy with teicoplanin i.m. At 7 weeks after inoculum, the femurs were explanted sterilely. The radiological score showed that the lowest and best radiological score was observed in Group 2 that was significantly different from the other groups. The highest bacterial load in the femoral canal was found in Group 1, which was significantly different from Group 2 and Group 4 (p < 0.05). Histology showed that Group 2 produced a marked improvement (p < 0.005) of the bone injuries induced by the osteomyelitis in comparison with the other groups (Smeltzer score). The current findings showed that tested device might effectively lead to MRSA infection healing after surgical debridement and immediate implantation.