Severe Acute Respiratory Infections (SARI) surveillance in over-65-years-old patients: the experience of a University hospital (seasons 2017-2018 and 2018-2019).

Background: Influenza is a relevant public health problem, also due to the risk of complications. The most effective measure to prevent influenza is vaccination; therefore, at present, there is consensus among European countries, regarding the need for routine seasonal influenza vaccination of elderly and individuals at increased risk of severe influenza. At the same time, influenza surveillance is necessary to understand the viruses circulating and effectiveness of vaccination strategies. The present study reports the results of two seasons influenza surveillance (2017/2018 and 2018/2019) conduced in an University Hospital in Rome among hospitalized patients aged ≥65 years. Study design: A prospective cohort study. Methods: The study consisted of systematic daily screening of all admissions among patients aged ≥65 years meeting a syndromic SARI case definition during two consecutive influenza seasons: 2017/2018 and 2018/2019. Characteristics of patients and their risk factors were collected by a standardized questionnaire and nose-pharyngeal swabs were performed to each patient. Influenza vaccine effectiveness (IVE), rates of vaccinated subjects and case fatality rate were also evaluated. Results: Influenza was laboratory confirmed in 11 (9.9%) of the 111 and 11 (9.6%) of the 115 enrolled patients in seasons 2017/18 and 2018/19, respectively. Adjusted IVE against all influenza type, calculated for each season, was 88.5% (95% CI: 38.9 to 97.8) and 61.7% (95% CI: -59.9 to 90.9) for 2017/2018 and 2018/2019 seasons, respectively. Our analysis shows a Case Fatality Rate of 2.7% and 4.3% for the 2017/18 and 2018/19 seasons, respectively. Conclusions: The surveillance of SARI conduced in one hospital in Rome confirmed that influenza is an important cause of hospital admissions. Routine monitoring of infectious diseases and related aetiology associated with SARI, also at the local-level, is useful for targeting the right preventive measures.

Surveillance of healthcare acquired infections by "alert microorganisms": preliminary results.

BACKGROUND: We evaluated the trend of four years (2015 - 2018) of "alert organisms" surveillance carried out at the 450 bed teaching hospital S. Andrea in Rome. METHODS: All patients with an "Alert organism" isolation were screened. In accordance with definitions used by the Centers for Disease Control patients with an "alert organism" isolation were evaluated for infection or colonization, by an infection control team (ICT). RESULTS: Between April 2015 and December 2018 a total 4,762 specimens with "Alert organism" isolation were screened and 1,601 patients were surveyed and included in the study. Overall 780 (48.8%) patients developed an healthcare acquired infection (HAI) at our institution, whereas 311 (19.4%) entered with a community acquired infection, 254 (15.8%) with an infection acquired in another healthcare setting and 256 (16.0%) resulted simply colonized. The 780 patients who developed an HAI at our institution presented 878 infectious episodes and the isolation of 931 microorganisms. C. difficile infections were the most common (27.2%), followed by 21.3% respiratory tract infections, 16.9% urinary tract infections, 15.5% surgical site infections, 12.5% bloodstream infections, 3.6% ulcers and 3.0% others. Among HAI group Gram negatives (54.1%) were more frequent than Gram positives (45.9%), whereas in patients entering in the hospital already with a community infection Gram positives overpassed Gram negatives (58.7% vs. 41.3%; p<0.001). Most common pathogens responsible for HAI were C. difficile (25.6%), Klebsiella spp. (25.5%), MRSA (19.6%) and Acinetobacter spp. (15.3%). Notably 30.0% HAI at other institutions were represented by C. difficile. Impressively, >40% of community acquired infections were related to MRSA. CONCLUSIONS: The present study provided some useful insight into the major multi-resistant pathogens epidemiology at our institution. The Authors succeeded in organizing a multidisciplinary ICT that created a partnership feeling with the hospital personnel.

Proteomics boosts translational and clinical microbiology.

The application of proteomics to translational and clinical microbiology is one of the most advanced frontiers in the management and control of infectious diseases and in the understanding of complex microbial systems within human fluids and districts. This new approach aims at providing, by dedicated bioinformatic pipelines, a thorough description of pathogen proteomes and their interactions within the context of human host ecosystems, revolutionizing the vision of infectious diseases in biomedicine and approaching new viewpoints in both diagnostic and clinical management of the patient. Indeed, in the last few years, many laboratories have matured a series of advanced proteomic applications, aiming at providing individual proteome charts of pathogens, with respect to their morph and/or cell life stages, antimicrobial or antimycotic resistance profiling, epidemiological dispersion. Herein, we aim at reviewing the current state-of-the-art on proteomic protocols designed and set-up for translational and diagnostic microbiological purposes, from axenic pathogens' characterization to microbiota ecosystems' full description. The final goal is to describe applications of the most common MALDI-TOF MS platforms to advanced diagnostic issues related to emerging infections, increasing of fastidious bacteria, and generation of patient-tailored phylotypes. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Microbial tracking of multidrug-resistant Klebsiella pneumoniae isolates in a pediatric hospital setting.

We investigated the clonal relatedness of seven multi-drug-resistant (MDR) Klebsiella pneumoniae isolates, as well as three susceptible K. pneumoniae isolates collected during hospital outbreaks and outbreak-related microbiological surveillance, respectively. The relatedness among K. pneumoniae isolates was assessed by pulsed field gel electrophoresis (PFGE) and automated repetitive-sequence-based PCR (rep-PCR) genotyping and the results were compared to a proteomic phenotyping performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All typing methods agreed on the generation of three different clusters of K. pneumoniae isogenetic/related MDR strains. After strengthening hospital infection control measures, no other spreading events involving MDR-K. pneumoniae were reported until the end of the observation period. This preliminary investigation suggests that, in a hierarchical approach to bacterial typing, MALDI-TOF MS proteome profiling might offer a fast and valuable preliminary screening tool able to support microbiologists during nosocomial outbreak surveys.

Evaluation by real-time PCR of the expression of S. flexneri virulence-associated genes ospB and phoN2 under different genetical backgrounds.

Under conditions of activated type III secretion Shigella flexneri up-regulates the expression of numerous genes, including the virulence plasmid (pINV)-encoded ospB and phoN2 genes. ospB and phoN2 are virulence-associated genes which are part of a bicistronic transcriptional unit encoding OspB, a protein (effector) of unknown function secreted by the type III secretion (TTS) apparatus, and PhoN2 (apyrase or ATP-diphosphohydrolase), a periplasmic protein involved in polar IcsA localization on the surface of S. flexneri. In this work we used real-time PCR to measure transcription of ospB and phoN2 of wild-type S. flexneri strain M90T as well as of derivative mutants impaired in definite virulence traits. The results obtained confirmed and extended previous reports indicating that the expression of ospB and phoN2 genes is modulated in a virB-dependent, mxiE-independent manner under conditions of non-activated secretion, while their expression is considerably induced in a mxiE-dependent manner under conditions of activated secretion. That the expression of the ospB-phoN2 operon is up-regulated in condition of activated secretion, indicates that probably the expression of these two genes might be important, especially during the later stages of infection of S. flexneri.

Evaluation of neutrophil CD64 expression and procalcitonin as useful markers in early diagnosis of sepsis.

Quantitation of neutrophil CD64 expression and procalcitonin (PCT) levels in blood samples have been recently proposed as useful tools for early detection of sepsis. To determine the usefulness of these tests, we analyzed blood samples of 112 patients, admitted to an intensive care unit (ICU), presenting clinical symptoms of sepsis, as well as of 50 healthy controls. At the end of the study, a retrospective analysis showed that only 52 of the 112 ICU-patients presented a real sepsis (positive blood culture). The results obtained indicated that of the 52 patients with sepsis, 50 and 49 presented levels of neutrophil CD64 expression >or= 2398 molecules per cell (cut-off determined by receiver operator characteristic analysis) and PCT levels >0.5 ng/ml (cut-off suggested by the manufacturer), respectively. However, the neutrophil CD64 test showed higher specificity in detecting sepsis since 5 out of the 60 ICU-patients without sepsis (negative blood culture), presented CD64 expression levels >or= 2398 molecules per cell, PCT levels >or= 0.5 ng/ml were shown in 27 patients. Moreover, while none of the 50 healthy controls presented a neutrophil CD64 level higher than the cut-off value, 5 patients presented PCT levels >or= 0.5 ng/ml. In conclusion, our data seem to indicate that the quantitation of CD64 expression could be taken into consideration as a sensitive and specific test for early diagnosis of sepsis.

Genotyping of different Pseudomonas aeruginosa morphotypes arising from the lower respiratory tract of a patient taken to an Intensive Care Unit.

Pseudomonas aeruginosa is an opportunistic pathogen and an ubiquitous environmental bacterium. Fifty-seven days after hospitalization, we isolated three distinct P. aeruginosa morphotypes (smooth, rough and mucoid) from the lower respiratory tract of a patient admitted to a Cardiology Intensive Care Unit (ICU). Moreover, a group of nine colony variants, arising from the three P. aeruginosa isolates growing in laboratory growth media, were also isolated. The resulting 12 isolates were characterised for antibiotic resistance profile and subjected to genotypic analysis by fluorescent-Amplified Fragment Length Polymorphism (f-AFLP) and automated repetitive extragenic palindromic-PCR (rep-PCR) fingerprinting. The three smooth, rough and mucoid morphotypes presented different antibiotic resistance profiles and genotyping analysis showed that they belonged to distinct clones, indicating that at day 57 after the admission the patient was simultaneously colonized by three distinct P. aeruginosa isolates. On the other hand, the nine colony variants presented heterogeneous antibiotic resistance profiles and clustered together with the three parental isolates. The understanding of the link between genotype plasticity and antibiotic resistance may contribute to improving our knowledge of this life-threatening pathogen.

Molecular characterization of virulence determinants of Stenotrophomonas maltophilia strains isolated from patients affected by cystic fibrosis.

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen which is currently isolated with increasing frequency from the airways of cystic fibrosis (CF) patients. In this study 13 S. maltophilia strains (11 isolated from the airways of independent CF patients, and two non-CF respiratory reference strains) have been characterized for the expression of several virulence-associated factors. In particular, the ability to form biofilm on abiotic surfaces has been determined and correlated with different features, such as motility, adherence and the ability to invade A549 respiratory epithelial cells. Moreover, the presence of a flagellum-associated gene as well as that of the StmPr1 gene, which encodes an extracellular protease, have been determined by Southern blot hybridization. Our data indicate that the different degree of biofilm formation exhibited by the 11 CF isolates does not correlate with motility, ability to adhere to and invade A549 cells, or with the presence of flagella. On the other hand, among the CF isolates the StmPr1 gene was found only in two strains, both able to establish chronic lung infections in CF patients. Moreover, only four of the strains analyzed show a temperature-independent antibiotic-resistance profile, suggesting either a different origin of these strains or an intervening adaptation to host tissues.

Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread.

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.

Chlamydia pneumoniae in PBMC: reproducibility of the OMPA nested touchdown PCR.

The aim of our study was to evaluate whether the replicate PCR testing may provide more accurate estimates of C. pneumoniae DNA prevalence in PBMC of patients undergoing carotid endarterectomy. Clinical sensitivity and reproducibility of ompA nested touchdown PCR was also performed. Clinical sensitivity and reproducibility was examined by testing C. pneumoniae-negative PBMC spiked with serial dilutions of semipurified C. pneumoniae elementary bodies (from 8 to 0.002 IFU/ml). Detection of C. pneumoniae DNA was performed by ompA nested touchdown PCR. Each clinical and spiked PBMC DNA specimen was analyzed in replicates of 1, 3, 5 and 10. PCR results of serial dilutions of C. pneumoniae DNA performed in replicates of 10 were analysed by probit analysis. C. pneumoniae DNA was detected in 14 of the 30 (46.7 %) PBMC clinical specimens examined when 10 replicates were tested. When we analyzed 1, 3 and 5 replicates, 4 (13.3 %), 7(23.3 %), 12(40 %) of the 30 specimens were positive, respectively. The limit of detection of ompA nested PCR touchdown was 0.008 IFU/ml when 10 replicates were tested. The ompA nested PCR had reproducibility scores of 10 for 10 from 8 to 4 IFU/ml concentration, but scores decreased for smaller numbers of IFU/ml. Our results showed that repeat testing of the same specimen increased clinical sensitivity as well as reproducibility of the ompA nested touchdown PCR. In conclusion the replicate PCR testing improves the performance of ompA nested touchdown PCR and provides a more accurate estimates of the prevalence of C. pneumoniae in PBMC of patients with atherosclerotic cardiovascular disease.

[Molecular diagnosis of Chlamydia pneumoniae diseases]. / Diagnostica molecolare delle infezioni da Chlamydia pneumoniae.

Chlamydia pneumoniae (C. p.) is an intracellular parasite directly involved in respiratory disease and more recently in chronic degenerative pathologies as atherosclerosis and asthma. Its peculiar life cycle makes cultural isolation difficult, thus, troublesome the diagnosis of the disease. Serology is so far the most common method of diagnosis of the, although the indirect based evidence of the serology may give clinically misleading results. Nucleic acid amplification methods offer indeed rapid, reliable and low cost assessment pathogen bacteria isolation, with relevant benefits for the patient's management. These molecular methods are nowadays essential in presence of bacteria of difficult cultivation or method inconsistent with temporal clinical needs, for they allow to rapidly detect even nucleic acid traces of the infectious agent, providing direct evidence of its presence in the biological samples and hence the relevant therapy. Nucleic acid methods are extensively applied in laboratory diagnosis of Chlamydia trachomatis bringing about the development of sensitive and reliable commercial kits. This review analyses the literature of the genic amplification methods in the search of C. p. in clinical samples highlighting methodological and diagnostic aspects. Although genic amplification methods have been implemented presently by the clinical research labs only, it is anticipated that through their standardisation they could be used by most clinical microbiology laboratories.

Community-acquired Acinetobacter radioresistens bacteremia in an HIV-positive patient.

We describe the first case of community-acquired bacteremia caused by Acinetobacter radioresistens; the patient was a 32-year-old HIV-positive neutropenic woman. Ambiguous Gram staining and poor biochemical reactivity of blood culture isolates misguided early diagnosis and therapy. Bacterial identification was based on 16S rDNA sequence analysis. A. radioresistens can be considered as a cause of opportunistic infection in immunodeficient patients.

Molecular characterization of first human Bartonella strain isolated in Italy.

The aim of this study was to characterize a Bartonella strain (BA-1) isolated from a blood culture of an Italian, human immunodeficiency virus-positive patient with bacillary angiomatosis. We analyzed the isolate using molecular biology methods such as whole-cell fatty acid analysis, PCR-restriction fragment length polymorphism analysis, type-specific 16S rRNA PCRs, sequence analysis of the 16S rRNA, pulsed-field gel electrophoresis, and arbitrarily primed PCR. The BA-1 isolate turned out to be a Bartonella quintana strain, similar but not identical to B. quintana Oklahoma, which was used as a control strain.

Travel-associated Burkholderia pseudomallei infection (Melioidosis) in a patient with cystic fibrosis: a case report.

In September 1997, a 25-year-old Italian woman with cystic fibrosis (CF) spent 3 weeks in Thailand. In August 1998, her pulmonary function rapidly declined, with productive cough and intermittent fever. Chest x-ray films revealed diffuse, small, patchy opacities in the upper lobes. Burkholderia pseudomallei (BP) was isolated from specimens of the patient's sputum and was identified by means of 16S rDNA sequencing. The diagnosis of melioidosis was serologically confirmed. Continuous therapy with ceftazidime and co-trimoxazole and maintenance with co-trimoxazole, doxycycline, and chloramphenicol resulted in eradication of BP. We present the issue of whether patients with CF represent a population particularly at risk for melioidosis.

Multiple-antibiotic resistance mediated by structurally related IncL/M plasmids carrying an extended-spectrum beta-lactamase gene and a class 1 integron.

A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extended-spectrum beta-lactamase gene, bla(SHV-5), was identified 3. 5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb bla(SHV-5)-In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expanded-spectrum cephalosporins and aminoglycosides.

Class 1 integron-borne multiple-antibiotic resistance carried by IncFI and IncL/M plasmids in Salmonella enterica serotype typhimurium.

The presence and genetic content of integrons were investigated for 37 epidemiologically unrelated multiple-drug-resistant strains of Salmonella enterica serotype Typhimurium from humans. All isolates were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and trimethoprim, as well as to tetracycline and/or nalidixic acid; 20% of them were also resistant to gentamicin and amikacin. Three different class 1 integrons (In-t1, In-t2, and In-t3) were identified by Southern blot hybridization, PCR, and DNA sequencing, and these integrons were found to carry the aadB, catB3, oxa1, aadA1a, aacA4, and aacC1 gene cassettes. Integrons In-t1 (aadB and catB3) and In-t2 (oxa1 and aadA1a) were both located on a conjugative IncFI plasmid of 140 kb. In-t3 (aacA4, aacC1, and aadAIa) was located on an IncL/M plasmid of 100 kb which was present, in association with the IncFI plasmid, in gentamicin- and amikacin-resistant isolates. Despite the extensive similarity at the level of the antibiotic resistance phenotype, integrons were not found on the prototypic IncFI plasmids carried by epidemic Salmonella strains isolated during the late 1970s. The recent appearance and the coexistence of multiple integrons on two conjugative plasmids in the same Salmonella isolate are examples of how mobile gene cassettes may contribute to the acquisition and dissemination of antibiotic resistance.