Usutu virus infections in humans: a retrospective analysis in the municipality of Modena, Italy.

OBJECTIVE: To monitor the spread and to evaluate the role for public health of Usutu virus (USUV) in an endemic area of Italy. METHODS: The survey was retrospectively conducted by detecting USUV RNA and USUV antibodies in cerebrospinal fluid and serum samples collected between 2008 and 2011 from 915 patients with or without neurologic impairments in the area of the municipality of Modena, Italy. Organs of birds and pools of mosquitoes were also tested for USUV RNA. Positive samples were partially sequenced and used for phylogenetic analysis. RESULTS: The presence of USUV RNA (1.1%; 95% confidence interval (CI) 0.6-2.0) was significantly (p <0.05) higher than that of West Nile virus (0%; 95% CI 0-0.33). USUV antibody level was 6.57% (95% CI 4.87-8.82), and it was significantly higher (p <0.05) compared to that of West Nile virus (p 2.96, 95% CI 1.89-4.62). Partial genome sequencing of USUV strains detected in humans, birds and mosquitoes revealed high nucleotide sequence identity within them and with the USUV strains isolated in Central Europe. CONCLUSIONS: USUV infection in humans is not a sporadic event in the studied area, and USUV neuroinvasiveness has been confirmed.

Multidrug-resistant tuberculosis outbreak in an Italian prison: tolerance of pyrazinamide plus levofloxacin prophylaxis and serial interferon gamma release assays.

The optimal treatment for latent tuberculosis infection (LTBI) in subjects exposed to multidrug-resistant (MDR) tuberculosis (TB) remains unclear, and the change in response of the QuantiFERON-TB Gold In-Tube (QTB-IT) test during and after treatment is unknown. Between May 2010 and August 2010, 39 prisoners at the 'Casa Circondariale' of Modena, Italy, were exposed to a patient with active pulmonary MDR TB. All contacts were tested with the tuberculin skin test and QTB-IT. Upon exclusion of active TB, subjects positive to both tests were offered 6 months' treatment with pyrazinamide (PZA) and levofloxacin (LVX). QTB-IT testing was repeated at 3 and 6 months after initial testing in all subjects who were offered LTBI treatment. Seventeen (43.5%) of 39 subjects tested positive to both tuberculin skin test and QTB-IT test, and 12 (70.5%) agreed to receive therapy with PZA and LVX at standard doses. Only five (41.6%) of 12 subjects completed 6 months' treatment. Reasons for discontinuation were asymptomatic hepatitis, gastritis and diarrhoea. The QTB-IT values decreased in all subjects who completed the treatment, in two (33%) of six of those who received treatment for less than 3 months and in one (50%) of two patients who discontinued therapy after 3 months. The QTB-IT test results never turned negative. Despite the small number of subjects, the study confirmed that PZA plus LVX is a poorly tolerated option for MDR LTBI treatment. We observed a large degree of variation in the results of the QTB-IT test results among participants. The study confirmed that the interferon gamma release assay is not a reliable tool for monitoring the treatment of MDR LTBI in clinical practice.

Changing Italian nosocomial-community trends and heteroresistance in Staphylococcus aureus from bacteremia and endocarditis.

Bloodstream infections due to Staphylococcus aureus (BSI) are serious infections both in hospitals and in the community, possibly leading to infective endocarditis (IE). The use of glycopeptides has been recently challenged by various forms of low-level resistance. This study evaluated the distribution of MSSA and MRSA isolates from BSI and IE in 4 Italian hospitals, their antibiotic susceptibility--focusing on the emergence of hVISA--and genotypic relationships. Our results demonstrate that the epidemiology of MRSA is changing versus different STs possessing features between community-acquired (CA)- and hospital-acquired (HA)-MRSA groups; furthermore, different MSSA isolated from BSI and IE were found, with the same backgrounds of the Italian CA-MRSA. The hVISA phenotype was very frequent (19.5%) and occurred more frequently in isolates from IE and in both the MSSA and MRSA strains. As expected, hVISA were detected in MRSA with vancomycin minimum inhibitory concentrations (MICs) of 1-2 mg/l, frequently associated with the major SCCmec I and II nosocomial clones; this phenotype was also detected in some MSSA strains. The few cases of MR-hVISA infections evaluated in our study demonstrated that 5 out of 9 patients (55%) receiving a glycopeptide, died. Future studies are required to validate these findings in terms of clinical impact.

Role of the quantiferon-TB test in ruling out pleural tuberculosis: a multi-centre study.

Diagnosing pleural tuberculosis (plTB) might be difficult due to limited sensitivity of conventional microbiology tools. As M. tuberculosis (MTB)-specific T cells are recruited into pleural space in plTB, their detection may provide useful clinical information. To this aim, in addition to standard diagnostic tests, we used the QuantiFERON-TB Gold In-Tube (QFT-IT) test in blood and pleural effusion (PE) samples from 48 patients with clinical suspicion of plTB, 18 (37.5%) of whom had confirmed plTB. Four of them (22.2%) tested positive with a nucleic acid amplification test for MTB. The tuberculin skin test was positive in most confirmed plTB cases (88.9%). Positive QFT-IT tests were significantly more frequent in patients with confirmed plTB, as compared to patients with an alternative diagnosis, both in blood (77.7 vs 36.6%, p=0.006) and in PE samples (83.3% vs 46.6%, p=0.02). In addition, both blood and PE MTB-stimulated IFN-gamma levels were significantly higher in plTB patients (p=0.03 and p=0.0049 vs non-plTB, respectively). In blood samples, QFT-IT had 77.8% sensitivity and 63.3% specificity, resulting in 56.0% positive (PPV) and 82.6% negative (NPV) predictive values. On PE, QFT-IT sensitivity was 83.3% and specificity 53.3% (PPV 51.7% and NPV 84.2%). The optimal AUC-derived cut-off for MTB-stimulated pleural IFN-gamma level was 3.01 IU/mL (77.8% sensitivity, 80% specificity, PPV 68.4% and NPV 82.8%). These data suggest that QFT-IT might have a role in ruling out plTB in clinical practice.

Interferon-gamma-release assays detect recent tuberculosis re-infection in elderly contacts.

The tuberculin skin test (TST) does not discriminate between recent and remote latent tuberculosis infection (LTBI). This study was carried out to test two interferon-gamma-based blood assays in recent contacts with high prevalence of remote LTBI. We performed a contact tracing investigation in a nursing home for the elderly, where elderly patients were exposed to a case of pulmonary tuberculosis. TST, QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB (TS.TB) were performed 8 weeks after the end of potential exposure. IFN-gamma measurements were recorded and correlation with exposure was evaluated. Twenty-seven (37.5%), 32 (44.4%) and 16 (22.2%) subjects were TST, TS.TB and QFT-G positive, respectively; agreement between TS.TB and QFT-G was good among exposed subjects only (K=0.915, 0.218 in unexposed, p<0.001). When amounts of IFN-gamma were corrected for the number of producing T cells, specific IFN-gamma production was significantly different between exposed and unexposed individuals (16.75+/-5.40 vs 2.33+/-0.71 IFN-gamma IU/1000 SFC, p=0.0001). QFT-G and TS.TB provided discordant results among elderly contacts. Unlike TST, the specific IFN-gamma response might discriminate between recent and long-lasting tuberculosis infection.

A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections.

The detection of specific serum antibodies is mainly achieved by enzyme-linked immunosorbent assay (ELISA). Here, we describe the setting up of a microarray-based serological assay to screen for IgG and IgM against vertically transmitted pathogens (Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus types 1 and 2, varicella zoster virus, Chlamydia trachomatis). The test, accommodated onto a restricted area of a microscope slide, consists of: (a) the immobilization of antigens and human IgG and IgM antibody dilution curves, laid down in an orderly manner; (b) addition of serum samples; (c) detection of antigen-serum antibodies complexes by indirect immunofluorescence. The IgG and IgM curves provide an internal calibration system for the interpolation of the signals from the single antigens. The test was optimized in terms of spotting conditions and processing protocol. The detection limit was 400 fg for the IgG assay and 40 fg for the IgM assay; the analytical specificity was >98%. The clinical sensitivity returned an average value of 78%, the clinical specificity was >96%, the predictive values were >73%, and the efficiency was >88%. The results obtained make this test a promising tool, suitable for introduction in the clinical diagnostic routine of vertically transmitted infections, in parallel (and in future as an alternative) to ELISA.

First human case of Usutu virus neuroinvasive infection, Italy, August-September 2009.

We report the first worldwide case of Usutu virus (USUV) neuroinvasive infection in a patient with diffuse large B cell lymphoma who presented with fever and neurological symptoms and was diagnosed with meningoencephalitits. The cerebrospinal fluid was positive for USUV, and USUV was also demonstrated in serum and plasma samples by RT-PCR and sequencing. Partial sequences of the premembrane and NS5 regions of the viral genome were similar to the USUV Vienna and Budapest isolates.

Gram-positive bloodstream infections in liver transplant recipients: incidence, risk factors, and impact on survival.

The objective of the study was to assess the incidence, risk factors, and survival of gram-positive bloodstream infections (GP-BSI(s)) among liver transplant recipients during the first year after transplantation. Between October 2000 and September 2006, 42 episodes of GP-BSI(s) occurred in 205 patients with an overall incidence of 0.20 episodes/patient. Coagulase-negative staphylococci were detected in 45.2% of cases, Enterococcus species in 42.9% (E faecalis, eight; E faecium, seven; E avium, two; E gallinarum, one) and Staphylococcus aureus in 11.9%. Retransplantation was the only independent risk factor for GP-BSI (odds ratio [OR], 0.253; 95% confidence interval (CI), 0.089 to 0.715; P = .009). Thirty-day mortality rate was 28.5% and S aureus infections were related to a poorer outcome. It is noteworthy that all the isolates of S aureus were methicillin-resistant. Ampicillin was inactive against all the strains of E faecium and 50% of E avium isolates, but active against all E faecalis and E gallinarum strains. All the isolates were glycopeptide-susceptible. No significant differences in mortality rate were observed in relation to sex, etiologies of end-stage liver disease, cytomegalovirus infection/reinfection, type of donor, rejection, or retransplantation. GP-BSI, the only independent risk factor for death (OR, 0.262; 95% CI, 0.106 to 0.643; P = .003), reduced the survival rate by 26% in the first year posttransplant. In conclusion, GP-BSI(s) impact significantly on morbidity and mortality posttransplant, particularly among retransplantations. Control measures are required to reduce the incidence of GP-BSI(s) in liver transplant recipients. These findings must be considered when empirical antimicrobial therapy is indicated while awaiting blood-culture results.

[Screening of latent tuberculosis infection in health care workers by QuantiFERON-TB and tuberculin skin test]. / Screening dell'infezione tubercolare latente in operatori sanitari con QuantiFERON-TB e test cutaneo tubercolinico.

UNLABELLED: Recent guidelines (MMWR 2005) recommend the use of QuantiFERON-TB (QFT-TB) as an alternative to the tuberculin skin test (TST) for the screening of latent tuberculosis infection (LTBI) in health care workers (HCWs). MATERIALS AND METHODS: 590 HCWs were screened for LTBI by TST and QFT-TB. Results were compared with risk factors for LTBI, determined by questionnaires. RESULTS: Both tests were significantly associated with non-Italian nationality [TCT (OR = 9.17), QFT-TB (OR = 3.65)], age > or = 45 years old [TCT (OR = 1.81), QFT-TB (OR = 2.36)], history of household contacts [TCT (OR = 2.65), QFT-TB (OR = 3.37], occupational exposure to tuberculosis (TB) patients [TCT (OR = 2.14), QFT-TB (OR = 1.93)]. 55 cases were discordant (28 QFT-TB-negatives/TCT-positives; 27 QFT-TB-positives/TCT-negatives). Both tests were not associated with workplace risk factors or TB risk level assessed in different hospital units. CONCLUSIONS: In HCWs employed in a low TB incidence area both QFT-TB and TCT were more associated with non occupational risk factors (nationality, age, household contacts) than with main determinants of workplace risk for LTBI.

Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital.

The epidemiological and antifungal susceptibility data for 94 episodes of candidaemia in an Italian tertiary-care hospital between January 2000 and August 2003 were evaluated by prospective laboratory-based surveillance. The incidence of fungaemia was 0.90 episodes/10 000 patient-days, and the most common species isolated were Candida albicans (40.4%), Candida parapsilosis (22.3%), Candida tropicalis (16.0%) and Candida glabrata (12.8%). Among 24 patients who received antifungal prophylaxis, non-albicans Candida spp. were more prevalent than C. albicans (p 0.012). The 30-day mortality rate was high (38.2%), particularly for haematological (71.4%) and solid-organ transplant patients (50.0%), and in individuals with C. tropicalis and C. glabrata bloodstream infections (60.0% and 50.0%, respectively). In-vitro susceptibility tests demonstrated that 95% of the isolates were susceptible to amphotericin B (MIC < 2 mg/L), 98.1% to posaconazole (MIC < 1 mg/L), 95.8% to flucytosine (MIC < 32 mg/L) and fluconazole (MIC < 64 mg/L), and 94.7% to itraconazole (MIC < 1 mg/L). Posaconazole was active (MIC 0.5 mg/L) against all three isolates of Candida krusei, which had reduced susceptibility to both fluconazole and itraconazole. Overall, non-albicans Candida spp. accounted for 60% of the episodes of candidaemia, which could be related to the use of antifungal prophylaxis. Resistance is still uncommon in Candida spp. recovered from blood cultures. The in-vitro activity of posaconazole is encouraging, and this agent could play an important role in the management of invasive candidiasis, including episodes caused by inherently less susceptible species such as C. krusei.

A simultaneous outbreak of Serratia marcescens and Klebsiella pneumoniae in a neonatal intensive care unit.

We describe two concurrent outbreaks of Serratia marcescens and Klebsiella pneumoniae in a neonatal intensive care unit (NICU). Over a 16-month period, a total of 27 infants were either colonized (N=14) or infected (N=13). There were 15 cases of S. marcescens and 11 cases of K. pneumoniae. Both micro-organisms were involved in one fatal case. Seven preterm babies developed septicaemia, two had bacteraemia, three had respiratory infections and one had purulent conjunctivitis. The S. marcescens and K. pneumoniae isolates were investigated by three molecular methods: enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR), arbitrary primed PCR with M13 primer, and random amplification of polymorphic DNA. Different patterns were found in the 16 S. marcescens epidemic isolates from 16 newborn infants. The major epidemic-involved genotype was linked to the first nine cases and this was subsequently replaced by different patterns. Eight different typing profiles were also determined for the 13 K. pneumoniae isolates from 12 newborn infants. Four K. pneumoniae bacteraemic strains proved to be identical. In conclusion, the typing results revealed that two different micro-organisms (S. marcescens and K. pneumoniae) were simultaneously involved in invasive nosocomial infections in preterm newborns. Two simultaneous clusters of cases were documented. Heterogeneous genotypes among both species were also demonstrated to be present in the NICU at the same time. A focal source for both micro-organisms was not identified but cross-transmission through handling was probably an important route in this outbreak. Strict adherence to handwashing policies, cohorting, isolation of colonized and infected patients, and rigorous environmental hygiene were crucial measures in the containment of the epidemic.

[Sepsis caused by Corynebacterium xerosis in neonatology: report of a clinical case]. / Sepsi da Corynebacterium xerosis in neonatologia: descrizione di un caso clinico.

The pathogenicity of the nondiphtheria corynebacteria, most commonly known as coryneform bacteria in humans has been recognized in the last two decades. Corynebacterium xerosis is part of the normal flora of the skin, nasopharynx, conjunctives and it has recently been isolated from vaginal swabs. During the last few years, there has been an increased number of case reports claiming an association of C. xerosis with diseases, like septicemia, endocarditis, pleuropneumonia, peritonitis, osteomyelitis, septic arthritis, mediastinitis, meningitis, ventriculitis specially in immunocompromised patients or surgical patients. Infections due to C. xerosis have been reported rarely in newborn. We report a case of sepsis due to C. xerosis in a newborn without evident immunodeficiency. Our case further support the recognition of C. xerosis as a human pathogen and reinforces the fact that it should not be routinely considered as a contaminant.

Prenatal diagnosis of congenital cytomegalovirus infection.

We report here the results of a study on the prenatal diagnosis of congenital cytomegalovirus (CMV) infection. The study was carried out by both PCR and virus isolation from amniotic fluid (AF) for 82 pregnant women at risk of transmitting CMV for the detection of (i) seroconversion to CMV immunoglobulin G (IgG) positivity during the first trimester of pregnancy, (ii) symptomatic CMV infection in the mother during the first trimester of pregnancy or intrauterine growth retardation detected by ultrasound or abnormal ultrasonographic findings suggestive of fetal infections, and (iii) seropositivity for CMV-specific IgM. For 50 women, fetal blood (FB) was also obtained and tests for antigenemia and PCR were performed. The results indicate that AF is better than FB for the prenatal diagnosis of CMV infection. PCR with AF has a sensitivity (SNS) of 100%, a specificity (SPE) of 83.3%, a positive predictive value (PPV) of 40%, and a negative predictive value (NPV) of 100%; rapid virus isolation with the same material has an SNS of 50%, an SPE of 100%, a PPV of 100%, and an NPV of 94.7%. Fewer than 10% of the women positive for IgM by enzyme immunoassay (EIA) had a congenitally infected fetus or newborn infant. When EIA IgM positivity was confirmed by Western blotting (WB) and the WB profile was considered, the percent transmission detected among women with an "at-risk" profile was higher than that observed among IgM-positive women and was the same as that among women who seroconverted during the first trimester of pregnancy (transmission rates of 29 and 25%, respectively).

Induction of apoptosis by Chlamydia psittaci and Chlamydia trachomatis infection in tissue culture cells.

The role of programmed cell death (apoptosis) in LLC-MK2 cells infected with Chlamydia trachomatis LGV2 serotype and Chlamydia psittaci 6BC strain was investigated using flow cytometry and TUNEL procedures. The number of apoptotic cells was significantly higher at 72 and 96 hours post infection in the Chlamydia infected cell cultures in comparison with mock-infected cells. We postulate the apoptotic process to be a mechanism induced by C. trachomatis and C. psittaci infection in LLC-MK2 cells.

Detection of serum antibodies against Chlamydia pneumoniae by in vitro neutralization and microimmunofluorescence assays.

Antibodies to Chlamydia pneumoniae (IOL-207) were studied by the microimmunofluorescence (MIF) assay and in vitro neutralization in serum samples from 230 healthy adults and 332 patients at risk of C. pneumoniae infection. In the MIF test, the prevalence of immunoglobulin G (IgG) antibody was 53.9% among healthy subjects and 40.7% in the patients. An MIF antibody titre of > or = 1 : 512, consistent with acute infection by C. pneumoniae was documented in 23 out of 332 patients, and in none of the healthy adults. C. pneumoniae complement-dependent neutralizing antibody was detected in 25.8% and 31.2%, respectively of MIF-positive sera from healthy subjects and patients. The neutralizing antibody detection rate was 52.2% among the 23 patients with MIF titres of > or = 1 : 512. Complement-independent neutralization was observed in only 5 sera from healthy subjects and in 3 sera from patients. The complement-dependent neutralizing ability of sera significantly (p < 0.001) correlated with MIF titres.

Detection of Chlamydia trachomatis by a ligase chain reaction amplification method.

BACKGROUND AND OBJECTIVES: The ligase chain reaction is an in vitro DNA amplification technique that exponentially amplifies selected DNA sequences. GOAL: To evaluate a ligase chain reaction assay for the detection of Chlamydia trachomatis cryptic plasmid DNA (LCx Chlamydia) in patients routinely attending a sexually transmitted disease center in Italy. STUDY DESIGN: Urethral or cervical swabs were obtained from 501 consecutive patients (334 men and 167 women). The samples were assayed in parallel with LCx Chlamydia and conventional tissue culture; discordant results were further assayed by direct immunofluorescence and a ligase chain reaction with alternate primers. RESULTS: After resolution of discordant results, the LCx method showed a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 99.3%, 96.7%, and 100% in men; 100%, 100%, 100%, and 100% in women; and 100%, 99.5%, 97.1%, and 100% overall, respectively. By comparison, the sensitivity of tissue culture was 81.4% in men, 50% in women, and 77.6% overall. CONCLUSIONS: The automated LCx method is sensitive, fast, and accurate and represents a useful diagnostic tool for C. trachomatis infection, even in low and medium prevalence populations.

An immunofluorescence assay in microwell plate (IFA) for detecting serum antibodies to Chlamydia pneumoniae.

An indirect immunofluorescence assay using a 96 microwell cell culture plate (IFA) was developed for the detection of antibodies to C. pneumoniae. The results obtained by IFA on 230 sera of healthy subjects were compared with those obtained with the microimmunofluorescence test. The correlation coefficient for IgG detection with the two methods was 0.96 showing good agreement.

Assessment of the "Amplicor" PCR test in the diagnosis of Chlamydia trachomatis infection.

We compared a commercially available PCR assay (Amplicor, Roche, Switzerland) and tissue culture isolation for the detection of C. trachomatis in urethral and/or endocervical swabs. Of the 200 patients studied (130 men and 70 women) PCR and tissue culture gave concordant results in 199 cases; in one case PCR was positive and culture negative. The Amplicor PCR assay proved fast and sensitive and suitable for routine use in most clinical microbiology laboratories.

In vitro activity of azithromycin against Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis in comparison with erythromycin, roxithromycin and minocycline.

The in vitro activity of azithromycin against 40 strains of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis was investigated in comparison with erythromycin, roxithromycin and minocycline. All C. trachomatis strains were inhibited by azithromycin at a concentration < or = 0.5 microgram/ml. The initial minimum inhibitory concentration (MIC) of the drug for U. urealyticum was 4 microgram/ml, whereas some resistance against the drug was shown by M. hominis. Erythromycin and roxithromycin presented almost comparable activities, whereas minocycline was slightly more active than macrolides against C. trachomatis (MIC < or = 0.25) and more active against M. hominis (initial MIC < or = 1 micrograms/ml). Only 97% of U. urealyticum strains were susceptible to 8 micrograms/ml of minocycline.