Predominance of Clostridium difficile 027 during a five-year period in Bolzano, Northern Italy.

Toxigenic Clostridium difficile is responsible for antibiotic-associated diarrhoea and other diseases. The increasing frequency and severity is attributed to highly-virulent ribotypes such as 027. The aim of the study was to collect epidemiological and molecular data for C. difficile isolates during 2009-2013 in the Central Hospital of Bolzano, Northern Italy. Stool samples from inpatients of the Bolzano Central Hospital were screened for toxins A and B, and C. difficile was cultured and tested for antibiotic susceptibility. PCRs were performed for genes of toxin A, toxin B, binary toxin and ribotyping. During the period 2009-13 from 320 patients (9% of patients tested) at least one stool sample proved positive for C. difficile toxins, and incidences for all hospital inpatients per 10,000 patient days (per 1,000 admissions) varied between 2.2 (1.5) and 4.3 (3.0). Out of 138 isolates (43% of total isolates were studied), 24 different ribotypes were identified. Isolates with ribotype 027 were predominant (38%), followed by 018 (13%) and 607 (10%). Whereas for ribotype 018 a significant decrease was seen during the five-year period, ribotype 027 increased significantly from 0% in 2009 to 64% in 2012, decreasing then to 10% in 2013. Isolates were sensitive to metronidazole and vancomycin, whereas isolates of the three major ribotypes were resistant to moxifloxacin. Our data indicates a significant change in C. difficile incidence rates and ribotype frequencies during the five-year period in the Central Hospital in Bolzano.

Use of Cidofovir for Cytomegalovirus Disease Refractory to Ganciclovir in Solid Organ Recipients.

BACKGROUND: Solid organ transplantation (SOT) frequently is complicated by cytomegalovirus (CMV) infections. Cidofovir (CDV) is active against CMV, including many ganciclovir (GCV)-resistant mutants, but often is considered to be too nephrotoxic for use after organ transplantation. PATIENTS AND METHODS: Seven males and two females (median age 50.1 years), including two kidney/pancreas, four lung, one small bowel, and two hand recipients, received CDV for refractory CMV disease. RESULTS: Three recipients were CMV seronegative, but all nine received grafts from CMV-seropositive donors. Five patients were given antithymocyte globulin, four received daclizumab induction, seven experienced rejection (five with multiple episodes), and one suffered from common variable immunodeficiency. Six presented with other infections (five invasive fungal and four bacterial). Eight patients had received prophylactic GCV, and eight had been treated for CMV infection/disease (GCV eight; CMV immunoglobulin three; foscarnet three). The indications for CDV were UL97 CMV mutation (n = 2), GCV-induced neutropenia with continued CMV disease (n = 4), and clinical resistance to GCV (n = 3). Seven patients cleared CMV, and two had a partial response. Four experienced CMV relapse requiring GCV (n = 2), repeat CDV (n = 1), or CMV immunoglobulin (n = 1). Four patients had mild nephrotoxicity, and three developed renal failure, all in association with additional factors. No patient died directly from CMV disease alone. Two patients died of uncontrolled infections and concurrent CMV disease, one with invasive aspergillosis and another with nocardiosis. CONCLUSIONS: Cidofovir was useful for the treatment of GCV-refractory CMV disease after SOT. Although nephrotoxicity was a common complication of CDV, several patients completed a course of therapy successfully and demonstrated effective treatment of CMV disease.

Long term complications following 54 consecutive lung transplants.

BACKGROUND: Due to the complex therapy and the required high level of immunosuppression, lung recipients are at high risk to develop many different long term complications. METHODS: From 1993-2000, a total of 54 lung transplantation (LuTx) were performed at our center. Complications, graft and patient survival of this cohort was retrospectively analyzed. RESULTS: One/five and ten-year patient survival was 71.4%, 41.2% and 25.4%; at last follow up (4/2010), twelve patients were alive. Of the 39 deceased patients, 26 died from infectious complications. Other causes of death were myocardial infarction (n=1), progressive graft failure (n=1), intracerebral bleeding (n=2), basilary vein thrombosis (n=1), pulmonary emboli (n=1), others (n=7). Surgical complication rate was 27.7% during the first year and 25% for the 12 long term survivors. Perioperative rejection rate was 35%, and 91.6% for the 12 patients currently alive. Infection incidence during first hospitalization was 79.6% (1.3 episodes per transplant) and 100% for long term survivors. Commonly isolated pathogens were cytomegalovirus (56.8%), Aspergillus (29.4%), RSV (13.7%). Other common complications were renal failure (56.8%), osteoporosis (54.9%), hypertension (45%), diabetes mellitus (19.6%). CONCLUSIONS: Infection and rejection remain the most common complications following LuTx with many other events to be considered.

Evaluation of a "home-made" method to determine viral genotype and characterize mutations conferring drug resistance in chronic hepatitis B patients.

We compared a home-made sequencing system to analyze plasma samples from patients with chronic HBV infection with the commercial TRUGENE(®) HBV Genotyping Assay. A PCR and sequencing protocol based on published primers was applied to detect the viral genotypes as well as the major patterns of point mutations leading to resistance to lamivudine, adefovir and entecavir. For the determination of HBV genotypes the obtained sequences were aligned with a database created within the RIDOM TraceEdit program and publicly available reference sequences. Our results showed perfect correlation with the commercial system, with types D (72%) and A (22%) being the most frequent genotypes. The resistance loci were also reliably detected with mostly combined L180M and M204V/I mutations as the local patterns. M204I mutations were more frequent in genotype D, M204V in genotype A isolates. G173L mutations were not found. The only genotype C isolate tested revealed a different pattern (E263D and I269L). These data speak for the usability of this rapid amplification and sequencing approach for routine genotyping of HBV isolates and simultaneous determination of the drug resistance profile of the dominant viral species.

High clonal heterogeneity of Panton-Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus strains from skin and soft-tissue infections in the Province of Bolzano, Northern Italy.

Panton-Valentine leukocidin (PVL)-positive community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to characterise 10 PVL-positive MRSA isolates collected during February 2010 to January 2011 from skin and soft-tissue infections in the North Italian Province of Bolzano. Accessory gene regulator (agr) typing, staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene typing, multilocus sequence typing, toxin gene profiling, polymerase chain reaction for type I arginine catabolic mobile element (ACME) and antimicrobial resistance typing were applied to the isolates. Eight different CA-MRSA clones were identified, including ST30-IVc, ST772-V, ST80-IVc, ST5-IVc, ST88-IVa, ST93-IVa, ST8-IVc and the type I ACME-positive ST8-IVa. The high heterogeneity of PVL-positive MRSA probably reflects the introduction of different clones by international travellers or immigrants.

Immunosurveillance for Mycobacterium tuberculosis of health care personnel in a third level care hospital.

BACKGROUND: Health care workers are at risk for Mycobacterium tuberculosis (MTB) infection. OBJECTIVES: To perform an occupational health survey among 621 employees of a 800-bed third level care hospital covered by MTB surveillance. METHODS: Statistical analysis was applied to results from tuberculin skin test (TST), QuantiFERON - TB Gold in tube assay (QFT), PPD-ELISA for serum antibodies, and occupational or vaccine data. RESULTS: 29.1% of subjects were TST positive, 18.5% were QFT positive. In 23% of subjects no correlation between these tests was found, presumably linked to BCG-vaccination, since TST positivity was 4 times higher among vaccinated subjects, whereas both tests correlated well in unvaccinated subjects. QFT values above 2 IU/ml were significantly associated with positive TST and age over 40 years. Working in MTB risk level 4 was significantly associated with QFT, TST and PPD-antibody levels, suggesting booster effects by repeated exposure. No clear correlation was observed with medical specializations but significantly higher QFTpositivity was found in subjects not assigned to the classical medical professions and originating from MTB high risk areas. CONCLUSIONS: These results shift the focus on maintenance personnel, who mostly worked in MTB risk level 2 areas. The less positive QFT results in vaccinated subjects highlight QFT's advantage as a screening tool and argue for a protective effect of the BCG-vaccine, although percentages of vaccinated persons varied largely between different medical professions. Interestingly, the percentage of QFT positive persons was lower among subjects reporting MTB exposure than those who were not aware of exposure events.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus from bacteraemia in northern Italy.

BACKGROUND: Vancomycin is frequently used in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia; reduced susceptibility to vancomycin is therefore disturbing. METHODS: molecular epidemiological analysis of 81 MRSA bacteraemia isolates collected during 2002-10 in the province of Bolzano, northern Italy was performed. MICs of a range of antimicrobials were determined by agar microdilution, screening for hGISA was by Macro-Etest and Etest GRD and confirmed by PAP-AUC with vancomycin and teicoplanin. All isolates were characterised by toxin gene profiling, agr, spa, and SCCmec-typing; MLST and PFGE were carried out on representative strains. RESULTS: The dominant clones identified were ST8-MRSA-IVc (55%) and ST228- and ST111-MRSA-I (25%); most of the latter two lineages (19/20; 95%) were GISA or PAP-AUC confirmed hGISA. One ST8-MRSA-IVc isolate harboured ccrA2B2 together with ccrA4B4. The remainder were diverse genotypically and belonged to MLST clonal complexes 1, 22, 45 and 398. CONCLUSIONS: Diverse lineages of MRSA were identified as causing bacteraemia in a province in northern Italy. The association of a specific genotype with the hGISA and GISA phenotypes among representatives of the second most common lineage identified is of clinical concern.

Investigation of bacterial and viral agents and immune status in Behcet's disease patients from Iran.

AIM: Behçet's disease (BD) is an autoimmune disorder associated with HLA-B51 positivity. Serologic/genomic findings have suggested microbes as possible causative agents and the geographical distribution suggests environmental influences. METHODS: We performed comparative analyses of 40 patients with BD or related symptoms not fulfilling BD criteria. Patients originating from different regions of Iran were tested by molecular/serological methods for human herpes viruses and parvovirus B19, two Chlamydiae species, as well as Coxiella, Listeria, Yersinia, Leptospira and Mycobacterium paratuberculosis. Human leukocyte antigen-typing was performed: testing of cytokine profiles and immune mediators representative for the cellular immune system, including neopterin/kynurenine production. RESULTS: No apparent differences in interleukin (IL)-4, 6, 8 and 10 were observed, whereas production of soluble IL-2-receptor and tumor necrosis factor (TNF)-alpha were more pronounced in the BD group. Neopterin/kynurenine production was comparable, although both groups showed twice the levels of healthy people. No significant differences of herpes simplex virus (HSV) antibody titres were observed but higher titres against Chlamydophila pneumoniae were found in the controls. In 20 BD patients and controls neither parvovirus B19 DNA was detected nor bacterial DNA. Viral DNA of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpes virus (HHV)8 was detected more frequently in the BD group, whereas HSV DNA was only found in the controls, indicating that stomatitis might be caused by HSV. CONCLUSION: Although no significant association of BD was detected with a single pathogen, our findings suggest that detection of HSV DNA or Chlamydiae would rather argue against classic BD. Whether there is a discriminative potential of the tested immune mediators/receptors has to be elucidated in further studies.

Dominance of CTX-M group 1 beta-lactamase enzymes in ESBL producing E. coli from outpatient urines in neighboring regions of Austria and Italy.

The importance of extended spectrum ß-lactamases (ESBL) is increasing worldwide. ESBLs of the CTX-M type are on the rise in Europe, not only in the hospital environment but also in outpatients. Therefore we performed a comparative pilot study including ESBL producing Escherichia coli isolated from outpatients suffering from urinary tract infections, 28 from Innsbruck, Austria, and 34 from Bolzano, Italy. Using established PCR methods we detected in nearly 90% of ESBL producing E. coli isolates CTX-M group 1 enzymes and only a few group 2 or group 9 enzymes. bla (TEM), bla (OXA-1) and aminoacyltransferase aac(6')-lb were significantly more frequent in the Austrian region, where also bla (SHV )was found in one isolate. In 2009 the overall prevalence of ESBL in E. coli causing urinary tract infection in outpatient samples was 7.6% in a local laboratory in Innsbruck and 5% in Bolzano. Additionally, we investigated plasmid-mediated qnr genes which can contribute to quinolone resistance, qnrA was found in an AmpC producing E. coli from Innsbruck and qnrS in two ESBL producers from Bolzano. Data confirmed that ESBL-producing E. coli have emerged as important pathogens in urinary tract infections of outpatients in both regions.

Complete nucleotide sequence of the IncN plasmid pKOX105 encoding VIM-1, QnrS1 and SHV-12 proteins in Enterobacteriaceae from Bolzano, Italy compared with IncN plasmids encoding KPC enzymes in the USA.

OBJECTIVES: We determined the complete nucleotide sequence of pKOX105, a 54 641 bp plasmid from a Klebsiella oxytoca strain that was isolated from a resident of a long-term-care facility in Bolzano, Italy. METHODS: The plasmid was sequenced using a shotgun approach. Combinatorial PCRs, directed PCRs and walking reads were used to assemble the contigs and to fill in gaps. Gene sequences were compared with reference plasmids and aligned with GenBank data using BLAST and CLUSTAL W software. RESULTS: pKOX105 belonged to incompatibility group IncN, harboured bla(VIM-1), bla(SHV-12), qnrS1, aacA4 and dfrA14 and conferred resistance to carbapenems, oxyimino-cephalosporins, quinolones, aminoglycosides and trimethoprim. It was highly related to the p9 and p12 plasmids from Klebsiella pneumoniae and K. oxytoca strains isolated at a New York City hospital in 2005 carrying bla(KPC-2) and bla(KPC-3), respectively. CONCLUSIONS: IncN plasmids are broad host-range plasmids that have contributed significantly to the worldwide dissemination of many different resistance genes in Enterobacteriaceae from animal and human sources. This plasmid family is now playing a crucial role in the global spread of diverse carbapenemase genes in Klebsiella spp.

Generalized parapoxvirus infection associated with increased antibody titres for varicella zoster virus and measles.

Human parapoxvirus infections are rare, self-limiting, zoonotic diseases. A 35-year-old veterinarian presented with a generalized rash of large umbilicated vesicles that appeared after antibiotic treatment for erysipelas on the forearm. The erysipelas arose from an erupted pustular thumb lesion that appeared after examining a sheep. An outbreak of chickenpox in the village suggested parapoxvirus or varicella zoster virus (VZV) was the most likely agent. No poxvirus was detected by electron microscopy or in cell cultures from lesion material. PCR revealed parapoxvirus DNA with a sequence similar to orf-viruses from Finland. Orf-virus immunofluorescence showed a titre increase, supporting the parapoxvirus diagnosis. VZV was not detected by PCR, but varicella antibodies increased three-fold in serum samples drawn two weeks apart. In addition, the patient had high antibody titres for measles and reported recent contact with individuals exposed to an outbreak of measles in nearby Austria. To explain the unusually generalized symptoms in this young and healthy patient, these findings could be variously interpreted as: i) a booster by community VZV infections; ii) a subclinical VZV (re)infection that was superinfected by the parapoxvirus; iii) an orf-virus mediated immune stimulation; iv) a post-infectious syndrome; or v) a temporary immunosuppression by subclinical measles.

Comparison of human metapneumovirus genotypes from the province of Bolzano in northern Italy with strains from surrounding regions in Italy and Austria.

The epidemiology of the genetic sublineages of human metapneumovirus (hMPV) and their clinical relevance are not fully understood. We compared hMPV genotypes isolated in the province of Bolzano in Northern Italy with strains from nearby Italian and Austrian regions by sequencing of NP- and L-gene fragments. Our results suggest that similar strains cycle through adjacent geographic areas, with the respective subtypes replacing each other on a seasonal basis.

Linkage of acquired quinolone resistance (qnrS1) and metallo-beta-lactamase (blaVIM-1) genes in multiple species of Enterobacteriaceae from Bolzano, Italy.

OBJECTIVES: Twenty-four of 209 oxyimino-cephalosporin- and/or aztreonam-resistant Enterobacteriaceae collected around Bolzano had reduced susceptibility or resistance to carbapenems and gave positive metallo-beta-lactamase (MBL) tests. Their resistance mechanisms were investigated. METHODS: Resistances were identified by Vitek 2 and MIC tests and isolates were genotyped by PFGE. Resistance genes were identified by PCR and sequencing, and plasmids were transferred by conjugation and/or transformation. Plasmid-borne genes were identified by Southern blotting, and their genetic surroundings were investigated by PCR mapping. RESULTS: The 24 isolates with positive EDTA/imipenem synergy tests had bla(VIM-1) carried on 40-150 kb plasmids. Imipenem MICs ranged from 2 to >32 mg/L, while those of meropenem and ertapenem were lower. The isolates included a clonal cluster of 10 Klebsiella pneumoniae, two other K. pneumoniae isolates, and diverse isolates of Escherichia coli (seven), Klebsiella oxytoca (three) and Citrobacter freundii (two). Six MBL producers were aztreonam-susceptible; the 18 aztreonam-resistant isolates had co-resident extended-spectrum beta-lactamases. bla(VIM-1) occurred as the first cassette in class 1 integrons, with aacA4 as the second cassette. Quinolone resistance gene qnrS1 was detected in 21 of 24 (87.5%) bla(VIM-1)-positive isolates versus 14 of 185 (7.6%) bla(VIM)-negative isolates (P < 0.0001), with 13 of the latter belonging to a clonal cluster of E. coli. qnrS1 was located on the same plasmids as bla(VIM-1) and aacA4, but was not closely linked, as judged by PCR mapping. CONCLUSIONS: bla(VIM-1) has become disseminated among enterobacteria in a small Italian town. The frequent association of genes conferring carbapenem, aminoglycoside and quinolone resistance on single plasmids will facilitate co-selection.

IFN-gamma mediated pathways in patients with fatigue and chronic active Epstein Barr virus-infection.

BACKGROUND: Chronic active Epstein Barr virus (EBV)-infection is characterized by mononucleosis like symptoms including fatigue, lymphadenopathy and/or hepatosplenomegaly and serologic evidence for ongoing EBV replication. Interferon-gamma (IFN-gamma) triggers several antiviral mechanisms in target cells including the induction of indoleamine-2,3-dioxygenase (IDO), which degrades the essential amino acid tryptophan to kynurenine. Because tryptophan is a precursor of the neurotransmitter 5-hydroxytryptamine (serotonin), tryptophan depletion by IDO can cause mood disturbances in patients with chronic immune activation. METHODS: This study investigated the tryptophan metabolism in 20 patients with chronic active EBV-infection, who were followed up for 4 to 8 months and in 10 healthy age-matched controls. The clinical suspicion of chronic active EBV infection was verified by the presence of circulating antibodies against EBV early antigen (EA) and virus capsid antigen (VCA). RESULTS: Patients with detectable EBV-DNA had higher serum neopterin (p<0.01) and lower tryptophan concentrations (p=0.01) than EBV-DNA negative patients. Serum concentrations of neopterin, indicating Th-1 mediated immune activation via IFN-gamma, were positively correlated to enhanced tryptophan degradation (rs=0.650, p<0.001) in patients, but not in healthy individuals. Patients suffering from more severe symptoms (as assessed by questionnaires) tended to have aggravated tryptophan degradation. CONCLUSION: Our data show that EBV viremia is associated with cell-mediated immune activation and increased tryptophan degradation, which may partly account for the symptoms found in this disorder.

Local administration of cidofovir for human papilloma virus associated skin lesions in transplant recipients.

Human papilloma virus (HPV)-associated diseases are increasingly diagnosed in solid organ recipients. Cidofovir (CDV) is a broad-spectrum antiviral agent with activity against all human herpes viruses and HPV. From 2000-2004, a total of 1303 solid organ transplants (SOT) were performed at our center. Six transplant recipients were treated with topical CDV for HPV-associated lesions. One cardiac recipient responded to a single injection of CDV into his recurrent anal condylomata. In a renal recipient with recurrent penile condylomata CDV was injected into the lesions four times (2 week interval) until lesions regressed. One renal recipient developed multiple vaginal and anal intradermal neoplasias, which relapsed after laser ablation. The lesions were repeatedly injected with CDV and completely disappeared. Two renal recipients with widespread verrucae vulgares were treated with CDV gel, which resulted in regression of the lesions. One patient developed donor derived verrucae vulgares on both transplanted hands, which responded to CDV gel. Four of the six patients were switched from calcineurin inhibitors (CNIs) to Sirolimus (SIR). CDV was found effective in the treatment of HPV-associated skin lesions in SOT recipients. It needs to be determined whether switch from CNIs to SIR might have contributed to the beneficial effect of CDV.

Human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology.

BACKGROUND: In immunocompromised patients, respiratory viruses are likely to lead to lower respiratory tract infections that cause severe morbidity and mortality. We conducted a prospective study from September 2003 to March 2004 to investigate the epidemiology and impact of human metapneumovirus (hMPV) on lung transplant recipients. METHODS: We collected 265 nasopharyngeal aspirates and bronchoalveolar lavages: 51 samples originated from immunocompromised adults, 49 from lung transplant recipients, and 2 from a bone marrow recipient. Additionally, 209 samples from hospitalized non-immunocompromised children and 5 samples from immunocompromised children were analyzed for replicating hMPV by a combined cell culture and reverse transcriptase polymerase chain reaction method that includes DNA sequencing of selected isolates. RESULTS: Twelve samples from lung transplant recipients (25%), 29 from non-immunocompromised children (14%), and 2 from a child with a renal transplant were positive for hMPV. Most of the cases clustered within 2 outbreaks in October/November and March. In immunocompromised patients, hMPV was isolated throughout the entire observation period. The same viral strains circulated in hospitalized children and in lung transplant recipients. A different strain was isolated during the interepidemic period, suggesting that hMPV infections may be transmitted among lung transplant recipients independently from the community outbreak situation. Clinical signs and symptoms varied from no symptoms to severe pneumonia or acute graft rejection. Significantly, the only deaths occurred in the hMPV-positive group. Of interest, identification of replicating hMPV significantly correlated with rejection symptoms present at the time point of sample collection. CONCLUSIONS: Results of the study suggest that hMPV may be added to the list of pathogens that are possibly associated with episodes of allograft rejection.

Functional T-cell anergy in a case of persistent polyclonal B-cell lymphocytosis.

The T-cell population of a patient with persistent polyclonal B-cell lymphocytosis (PPBL) presenting with an intermittent Epstein-Barr virus (EBV)-associated disease was studied. Unstimulated T-cells did not express CD40 ligand (CD40L), whereas activation with IL-2 led to expression of this costimulatory molecule. CD40L expression was inhibited upon incubation with the supernatant of an EBV-positive B-cell line (SM) which had been grown spontaneously from the patient's peripheral blood cells. The supernatant of SM cells effectively inhibited cytotoxic T-cells. Elevated levels of IL-10, TNF-alpha and soluble CD40 were found in the supernatant of SM cells. Additionally, enhanced levels of LMP-1 protein were detected.

Tolerability and efficacy of N-chlorotaurine in epidemic keratoconjunctivitis--a double-blind, randomized, phase-2 clinical trial.

The aim of this study was to assess the tolerability and efficacy of N-chlorotaurine (NCT), an endogenous antimicrobial agent, in epidemic keratoconjunctivitis. In a prospective double-blind, randomized phase 2b study, the infected eyes were treated for 7 days with eye drops containing 1% aqueous solution of N-chlorotaurine (33 subjects) or gentamicin (27 subjects, control group). Adenovirus types 3, 4, 8, 19, and 37 were detected in 39 subjects (65%), enteroviruses in 8 (13.3%), and staphylococci in 5 (8.3%). Subjective and objective symptoms were scaled and added to a subjective and objective score, respectively, on day 1 (baseline), day 4, and day 8. Analyzing the whole study population, the subjective score on day 8 was lower in the NCT group (P = 0.016), whereas there were no differences in the objective score. However, in severe infections caused by adenovirus type 8 (n = 20) both the subjective and objective score were lower in the NCT group on day 4 (P = 0.003 and 0.015, respectively), which was also true for the subjective score on day 8 (P = 0.004) in this subgroup. The frequency of subepithelial infiltrates was similar in both groups. N-chlorotaurine was well-tolerated, shortened the duration of illness, and seems to be a useful causative therapeutic approach in severe epidemic keratoconjunctivitis.