Phylogenetic analysis of West Nile virus isolates, Italy, 2008-2009.

To determine the lineage of West Nile virus that caused outbreaks in Italy in 2008 and 2009, several West Nile virus strains were isolated from human specimens and sequenced. On the basis of phylogenetic analyses, the strains isolated constitute a distinct group within the western Mediterranean cluster.

Absence of neuroinvasive disease in a liver transplant recipient who acquired West Nile virus (WNV) infection from the organ donor and who received WNV antibodies prophylactically.

We describe the first case of West Nile virus (WNV) infection in Europe with transmission from donor to recipient following liver transplantation. The infection was detected in the recipient 3 days after transplantation, during the asymptomatic phase. We also report an innovative prophylactic strategy based on infusion of WNV hyperimmune plasma and gamma globulins that could be effective in preventing the appearance of a neuroinvasive disease.

Multiple nucleotide substitutions in the Neisseria meningitidis serogroup C ctrA gene cause false-negative detection by real-time PCR.

Two strains of Neisseria meningitidis serogroup C with disparate sequences of ctrA were isolated. The nucleotide substitutions did not alter the corresponding protein sequences, but they impeded the detection of these meningococcal isolates by real-time PCR.

False-positive transcription-mediated amplification assay detection of West Nile virus in blood from a patient with viremia caused by an Usutu virus infection.

Detection of West Nile virus (WNV) by nucleic acid amplification technology (NAAT) is used widely to screen blood and organ donations in areas where WNV is endemic. We report a false-positive result of a WNV transcription-mediated amplification assay (TMA) in a patient with viremia that was caused by Usutu virus, a mosquito-borne flavivirus.

Identification of an invasive infection of R. oryzae in a haematological patient using a molecular technique.

We report a case of cerebral mucormycosis in a 28-year-old male who was affected by chronic myeloid leukaemia and underwent allogeneic bone marrow transplantation. Nine months post-transplantation, he was admitted to the hospital with fever, bilateral eyelid oedema and neutropenia. X-ray analysis showed numerous areas of pulmonary parenchymal thickening, and a computed tomography scan of the brain showed inflammation of the frontal, maxillary, ethmoidal and sphenoidal sinuses and diffuse swelling of the periorbital tissues. Sinus cultures were taken, and based on its characteristic rhizoid structure, we classified the isolated fungus as a member of the genus Rhizopus. The fungus was identified as an Rhizopus oryzae species, as assessed by sequencing of the internal transcribed spacer of the rRNA gene. Treatment with amphotericin B was ineffective, however, and the patient died 2 weeks after admission. This case highlights the potential severity of an invasive infection of R. oryzae, identified by molecular biology techniques.

Detection of Treponema denticola in root canal systems in primary and secondary endodontic infections. A correlation with clinical symptoms.

AIM: The aim of the study was to investigate the presence of Treponema denticola in primary and secondary root-infected canal systems with periapical pathology and correlations with clinical signs and symptoms. METHODOLOGY: Endodontic samples were obtained from canals of 102 teeth: 79 had primary endodontic disease and 23 secondary endodontic disease. For each tooth, clinical data including symptoms and X-ray appearance were examined. The presence of T. denticola biological samples from the root canal space was detected by a PCR assay. RESULTS: T. denticola was detected in 24 out of the 79 teeth with primary infection and in 8 out of the 23 teeth with secondary infection. Teeth with specific clinical symptoms were frequently associated with T. denticola presence inside the root canal system. CONCLUSIONS: The presence of T. denticola in root canal system in association with specific clinical signs and symptoms of endodontic disease strongly suggests that this spirochete might play a critical role in the pathogenesis of the acute infection and rapid bone tissue alterations in both primary and secondary endodontic infections.

Recovery of Enterococcus faecalis in root canal lumen of patients with primary and secondary endodontic lesions.

The presence of Enterococcus faecalis in root canal teeth affected by primary and secondary periapical lesions was studied using polymerase chain reaction (PCR) assays. The association between presence of E. faecalis with clinical signs of apical lesions was assessed to evaluate a possible relationship between clinical findings. Microbial samples were obtained from healthy patients affected by different periapical lesions, 79 teeth with primary periapical lesion and 23 with secondary periapical lesion. For each tooth, clinical symptoms and X-ray appearance were examined. E. faecalis was detected in 6 of 79 samples with primary lesion (7.6%), and in 9 of 23 with secondary lesion (39.1%). Suggested association was found between E. faecalis and secondary apical lesions. As regard specific signs and symptoms E. faecalis was more associated with asymptomatic lesions (all p<0.05) than with symptomatic apical lesions. The study confirms the high presence of E. faecalis in secondary apical lesions. However, its effective role in endodontic pathogenesis such as bone periapical lesions needs to be clarified.

Late-onset group B streptococcal disease by infected mother's milk detected by polymerase chain reaction.

Late-onset Group B streptococcal (GBS) disease is a cause of illness, death and neurological sequelae in infancy. The epidemiology and pathogenesis of late-onset GBS disease is poorly defined. Infected breast-milk has been suggested as a source of postnatal infection and invasive disease. We describe a late-onset GBS disease by infected mother's milk in a term newborn in which the detection of GBS in neonatal bloodstream (confirmed by culture) and in the mother's milk was performed by PCR.

Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection.

AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C. pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes. METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-alpha by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzyme-linked immunosorbent assay. RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20. The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture. Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-alpha levels. CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis.

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorferi.

AIM: To evaluate the production of reactive oxygen species (ROS) and the expression of inducible nitric oxide synthase (iNOS) in rat isolated Kupffer cells (KCs) stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS: Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Purified Kupffer cells were tested in vitro with alive L. interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS: B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION: Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.

Comparative evaluation of three different ELISA methods for the diagnosis of early culture-confirmed Lyme disease in Italy.

In this study the raising and development of the immune response to Borrelia burgdorferi infection in 45 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans was investigated. A total of 95 serially collected serum samples were tested by using three different commercial ELISAs: recomWell Borrelia (Mikrogen), Enzygnost Borreliosis (DADE Behring) and Quick ELISA C6 Borrelia (Immunetics). The sensitivities of the ELISAs were as follows: Enzygnost Borreliosis IgM, 70.5 %; Quick ELISA C6 Borrelia, 62.1 %; recomWell Borrelia IgM, 55.7 %; recomWell Borrelia IgG, 57.9 %; and Enzygnost Borreliosis IgG, 36.8 %. In order to compare the specificity values of the three ELISAs, a panel of sera obtained from blood donors (210 samples coming from a non-endemic area and 24 samples from an endemic area) was tested, as well as sera from patients suffering from some of the most common biological conditions that could result in false-positive reactivity in Lyme disease serology (n = 40). RecomWell Borrelia IgG and recomWell Borrelia IgM were the most specific (97.1 % and 98.9 %, respectively), followed by Quick ELISA C6 Borrelia (96.7 %). Enzygnost Borreliosis IgG and IgM achieved 90.1 % and 92.3 % specificity, respectively. Sera that gave discrepant results when tested by the three ELISAs were further analysed by Western blotting.

Molecular detection of Treponema denticola and Porphyromonas gingivalis in carotid and aortic atheromatous plaques by FISH: report of two cases.

Treponema denticola and Porphyromonas gingivalis have been identified in atheromatous plaques of two patients suffering from atherosclerosis by PCR and fluorescence in situ hybridization (FISH). The use of the FISH technique suggested that these periodontopathic micro-organisms might be metabolically active within the wall of arteries, under the atherosclerotic lesion.

Comparative evaluation of two enzyme linked immunosorbent assay methods and three Western Blot methods for the diagnosis of culture-confirmed early Lyme borreliosis in Italy.

This study investigated the onset and development of the immune response to Borrelia burgdorferi infection in 30 Italian patients with culture-confirmed Lyme Borreliosis in the stage of erythema migrans (EM). All patients received antimicrobial treatment when entering the study and were prospectively evaluated monthly for up to 30 days after enrolment. A total of 60 serially collected serum samples were tested by using two different commercial enzyme-linked immunosorbent assays (ELISAs): Anti-Borrelia plus VlsE ELISA, Euroimmun, and the synthetic peptide-based ELISA, Quick ELISA C6, Immunetics. Sixty-five potentially cross-reacting sera were also tested. Anti-Borrelia plus VlsE ELISA IgG was far more sensitive than Quick ELISA C6 (56.6% and 33.3%, respectively). Moreover, considering that 17 additional sera from the first bleeding group of Lyme disease patients were IgM positive when tested by Anti-Borrelia plus VlsE IgM, the sensitivity of Anti-Borrelia plus VlsE as a whole system rose to 85.0%. Nevertheless, due to the specificity values of Anti-Borrelia plus VlsE ELISA identified in this study (98.5% for IgG and 78.5% for IgM), the need of a confirmatory test for the diagnosis of Lyme disease remains. All the sera were also tested by two different commercial Western Blot (WB) assays: Euroline-WB against Borrelia, Euroimmun, and Qualicode B. burgdorferi WB, Immunetics, in comparison with a multispecies "home made" WB. Performances of the three WB methods for the detection of IgM were very similar. On the contrary, these WBs performed with different values of sensitivity and specificity when IgGs were evaluated. The most sensitive method was the "home-made" WB IgG (71.7%), followed by the Euroline-WB IgG against Borrelia (68.3%). Qualicode B. burgdorferi WB IgG demonstrated to be only 26.6% sensitive. Both "home-made" WB IgG and Qualicode B. burgdorferi WB IgG were 100% specific, whereas Euroline-WB IgG against Borrelia scored 12 cross-reacting samples as borderline, showing a specificity value of 80.0%.

Treponema denticola infection is not a cause of false positive Treponema pallidum serology.

It has long been assumed that parodontal disease can be a cause of false positive results in syphilis serology, but so far there are no definitive data supporting this hypothesis. In this study we tested 250 serum samples obtained from blood donors. All of them were negative when routinely screened for antibodies against Treponema pallidum. Then, all these samples were tested by immunoenzymatic (ELISA) and Western Blot (WB) assays to investigate reactivities against T. denticola. Thirteen samples showed a strong positivity when tested by both methods. When tested by WB against T. pallidum no sample met the positivity criteria. Nevertheless, bands with molecolar weights of about 30-35 KDa (endoflagellar core antigens) were recognized. All the 13 subjects serologically T. denticola positive underwent oral clinical and radiological observation: all showed a very poor parodontal status (CPSS > 103). Eleven crevicular fluid samples out of the total of 13 patients were T. denticola positive by Real Time PCR carried out using a LightCycler system. In this study we demonstrated that the presence of T. denticola in the crevicular fluid samples obtained from patients with a severe periodontal status and/or a positive serology against T. denticola is not a cause of false positive results in syphilis serology.

Investigation of the antimicrobial activity at safe levels for eukaryotic cells of a low power atmospheric pressure inductively coupled plasma source.

Low power atmospheric pressure inductively coupled thermal plasma sources integrated with a quenching device (cold ICP) for the efficient production of biologically active agents have been recently developed for potential biomedical applications. In the present work, in vitro experiments aimed at assessing the decontamination potential of a cold ICP source were carried out on bacteria typically associated with chronic wounds and designed to represent a realistic wound environment; further in vitro experiments were performed to investigate the effects of plasma-irradiated physiological saline solution on eukaryotic cells viability. A thorough characterization of the plasma source and process, for what concerns ultraviolet (UV) radiation and nitric oxide production as well as the variation of pH and the generation of nitrates and nitrites in the treated liquid media, was carried out to garner fundamental insights that could help the interpretation of biological experiments. Direct plasma treatment of bacterial cells, performed at safe level of UV radiation, induces a relevant decontamination, both on agar plate and in physiological saline solution, after just 2 min of treatment. Furthermore, the indirect treatment of eukaryotic cells, carried out by covering them with physiological saline solution irradiated by plasma, in the same conditions selected for the direct treatment of bacterial cells does not show any noticeable adverse effect to their viability. Some considerations regarding the role of the UV radiation on the decontamination potential of bacterial cells and the viability of the eukaryotic ones will be presented. Moreover, the effects of pH variation, nitrate and nitrite concentrations of the plasma-irradiated physiological saline solution on the decontamination of bacterial suspension and on the viability of eukaryotic cells subjected to the indirect treatment will be discussed. The obtained results will be used to optimize the design of the ICP source for an effective production of reactive species, while keeping effluent temperature and UV radiation at values compatible with biomedical treatments.

Impact of meningococcal C conjugate vaccination campaign in Emilia-Romagna, Italy.

The incidence of reported meningococcal disease in Italy is among the lowest in Europe. The trend of the disease was increasing up to 2005 and then declined after the gradual introduction of a universal Men C vaccination program in 17/21 Italian regions. Since 2006, in Emilia-Romagna region vaccination against Neisseria meningitidis serogroup C was actively offered free of charge in a single dose to the age groups 12-15 months and 14-15 years, in addition to people with defined epidemiological risk. Our aim was to measure the impact of vaccination on the incidence of meningococcal disease caused by different serogroups among the population of Emilia Romagna Region, Northern Italy (approximately 4.5 million inhabitants) subdivided by age. Using surveillance data, we computed the incidence rates of Neisseria meninigitidis related invasive disease per 100.000 inhabitants for the years 2000 to 2012. In addition, the percentage change in incidence and the mortality rates were calculated. Results indicate a 70.1% decrease in the incidence of meningococcus C-related invasive disease after the introduction of MenC universal vaccination. No case of serogroup C related infection was observed since 2006 in children aged 1-4 years. These findings suggest that the single-dose vaccination strategy against serogroup C N.meningitidis targeted to the age groups 12-15 months and 14-15 years was effective in the Emilia-Romagna population. However, the occurrence of two cases of meningiditis in a 5-month child and in a 9-years child suggests caution and careful consideration in surveillance for the next years.

Comparative genomic and phylogenetic analysis of the first Usutu virus isolate from a human patient presenting with neurological symptoms.

Usutu virus (USUV) is a mosquito-borne flavivirus, belonging to the Japanese encephalitis antigenic complex, that circulates among mosquitoes and birds. We describe and analyze the complete genome sequence of the first USUV strain isolated from an immunocompromised patient with neuroinvasive disease. This USUV isolate showed an overall nucleotide identity of 99% and 96%, respectively, with the genomes of isolates from Europe and Africa. Comparison of the human USUV complete polyprotein sequence with bird-derived strains, showed two unique amino acid substitutions. In particular, one substitution (S595G) was situated in the DIII domain of the viral Envelope protein that is recognized by flavivirus neutralizing antibodies. An additional amino acid substitution (D3425E) was identified in the RNA-dependent RNA polymerase (RdRp) domain of the NS5 protein. This substitution is remarkable since E3425 is highly conserved among the other USUV isolates that were not associated with human infection. However, a similar substitution was observed in Japanese encephalitis and in West Nile viruses isolated from humans. Phylogenetic analysis of the human USUV strain revealed a close relationship with an Italian strain isolated in 2009. Analysis of synonymous nucleotide substitutions (SNSs) among the different USUV genomes showed a specific evolutionary divergence among different countries. In addition, 15 SNSs were identified as unique in the human isolate. We also identified four specific nucleotide substitutions in the 5' and 3' untranslated regions (UTRs) in the human isolate that were not present in the other USUV sequences. Our analyses provide the basis for further experimental studies aimed at defining the effective role of these mutations in the USUV genome, their potential role in the development of viral variants pathogenic for humans and their evolution and dispersal out of Africa.

Diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies.

West Nile virus, genus Flavivirus, is transmitted between birds and occasionally other animals by ornithophilic mosquitoes. This virus also infects humans causing asymptomatic infections in about 85% of cases and <1% of clinical cases progress to severe neuroinvasive disease. The virus also presents a threat since most infections remain unapparent. However, the virus contained in blood and organs from asymptomatically infected donors can be transmitted to recipients of these infectious tissues. This paper reviews the presently available methods to achieve the laboratory diagnosis of West Nile virus infections in humans, discussing the most prominent advantages and disadvantages of each in light of the results obtained during four different External Quality Assessment studies carried out by the European Network for 'Imported' Viral Diseases (ENIVD).