Comparison of nested PCR and real time PCR of Herpesvirus infections of central nervous system in HIV patients

BACKGROUND: Molecular detection of herpesviruses DNA is considered as the reference standard assay for diagnosis of central nervous system infections. In this study nested PCR and real time PCR techniques for detection of Herpes simplex virus type 1 (HSV-1), Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in cerebrospinal fluid of HIV patients were compared. METHODS: Forty-six, 85 and 145 samples previously resulted positive for HSV-1, CMV and EBV by nested PCR and 150 randomly chosen negative samples among 1181 collected in the period 1996–2003 were retrospectively reassessed in duplicate by real time PCR and nested PCR. RESULTS: Samples giving positive results for CMV, HSV-1 and EBV with nested PCR were positive also with real time PCR. One of the negative samples resulted positive for HSV and one for EBV. Real time PCR showed comparable sensitivity and specificity vs nested PCR. CONCLUSION: Real time PCR proved to be a suitable method for diagnosis of herpesvirus infections in CNS, showing comparable sensitivity and being less time consuming than nested PCR

Diffusion and transmission of carbapenem-resistant Klebsiella pneumoniae in the medical and surgical wards of a university hospital in Milan, Italy

Summary: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is emerging as a public health problem worldwide. In Italy, a remarkable increase in CRKP cases has been reported since 2010. In this study, CRKP diffusion, distribution and in-hospital transmission trends were evaluated in a university hospital in Milan, Italy, from January 2012 to December 2013. Isolates from 63 newly detected CRKP-positive patients were genotyped, and possible transmission was determined by combining the molecular results with data concerning the patients' admission and in-hospital transfers. Most of the cases (90.4%) were from general medical and surgery wards, and the remaining 9.6% were from the intensive care unit. Fifteen of the 46 hospital-associated cases (32.6%) were attributable to in-hospital transmission. After the introduction of targeted and hospital-wide control measures, the transmission index significantly decreased from 0.65 to 0.13 (p = 0.01). There was also a decrease in the overall nosocomial case incidence, from 0.37 to 0.17 per 1000 person-days (p = 0.07).Our findings indicate that the spread of CRKP in Northern Italy hospitals may go far beyond high-risk settings (i.e., intensive care units) and that strict surveillance should be extended to general areas of care. Keywords: Multidrug-resistant agents, Carbapenem-resistant Klebsiella pneumoniae, Cross-transmission, Infections control measures, Active surveillance, Active screenin

COVID-19 disease in clinical setting: impact on gonadal function, transmission risk, and sperm quality in young males.

Abstract Objectives We want to evaluate the possible presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in semen samples and semen quality, looking for a possible relationship between the infectious disease and fertility. Methods In this prospective study, we enrolled 15 consecutive men (age 18–50 years) with positive oropharyngeal swab to SARS-CoV-2 and classified, according to WHO criteria, in mild to moderate disease. A semen sample was collected to detect SARS-CoV viral RNA by the automated Real-Time PCR ELITe InGenius® system and the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup, France). Analysis of semen characteristics was performed according to WHO laboratory manual 5th ed. for the examination and processing of human semen. Blood samples for the dosage of hormonal assay, procalcitonin, interleukin 6, C-reactive protein were obtained. Results SARS-CoV-2 RNA has not been detected in semen samples from any of the subjects analysed. Sperm analysis exhibited abnormal seminal values in 14 out of 15 patients (93.3%). Furthermore, no difference was detected regarding sperm quality between mild and moderate SARS-CoV-2 patients. No alteration in the inflammatory indices was observed in the studied population, as well gonadotropins and testosterone levels. Conclusions COVID patients studied exhibits alteration of the seminal fluid both in microscopic and macroscopic characteristics such as hypoposia and increased viscosity, which have not been detected in previous studies. The presence of viral RNA within the seminal fluid was excluded

Detection of ST1702 Escherichia coli blaNDM-5 and blaCMY-42 genes positive isolates from a Northern Italian hospital

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy

Imported Loa Loa Filariasis: Three Cases and a Review of Cases Reported in Non-Endemic Countries in the Past 25 Years

Summary Objectives The aim of this study was to highlight the increasing chance of Western physicians encountering patients (both immigrants and expatriates/travelers) seeking help for loiasis. Methods We describe three cases of imported loiasis observed at two hospitals in Italy and France, and present a review of all previously published cases in the medical literature in the last 25 years (1986–2011). The search was performed using PubMed and Scopus databases using the terms " Loa loa " AND "loiasis". Results We reviewed 101 cases of imported loiasis of which 61 (60.4%) were reported from Europe and 31 (30.7%) from the USA. Seventy-five percent of infestations were acquired in three countries: Cameroon, Nigeria, and Gabon. Overall, peripheral blood microfilariae were detected in 61.4% of patients, eosinophilia in 82.1%, eye worm migration in 53.5%, and Calabar swellings in 41.6%. However, Calabar swellings and eosinophilia were more common among expatriates/travelers, whereas African immigrants were more likely to have microfilaremia. Eye worm migration was observed in a similar proportion in the two groups. Only 35 patients (including the three described here) underwent clinical follow-up for a median period of 10.5 months (range 1–84 months); clinical relapse occurred in three of these patients and persistence or reappearance of blood microfilaria in another two. Conclusions Due to increasing travel and the migration of people from the endemic countries of West Africa to Europe and the USA, we speculate on the possible emergence of loiasis. Western physicians should be aware of the typical (eye worm migration and Calabar swellings) as well as unusual clinical presentations

Influenza A/H1N1/2009 virus - experience of the clinical microbiology laboratory of the "L. Sacco" University Hospital in Milan

In the spring of 2009, a new variant of influenza A/H1N1 virus that had never been isolated before, was identified. From April 27 to December 31, 2009 the respiratory samples of 974 patients, obtained from suspected cases of pandemic influenza A virus infection, were analyzed at the Clinical Microbiology Laboratory of the "L. Sacco" University Hospital in Milan. The diagnosis of influenza A/H1N1 infection was performed initially through the use of different molecular biological methods: Seeplex® RV12 ACE Detection (Seegene), NUCLISENS® EASYQ® INFLUENZA A/B (bioMérieux), Influenza A/B Q-PCR Alert (Nanogen) running in parallel with rRT-PCR (CDC) to confirm the positivity to the new influenza virus, then was used a single specific test, Fast set H1N1v (Arrow Diagnostics). Retrospective study of data showed that 293 (30.1%) patients were positive for the new strain of influenza A/H1N1 virus and 8 (0.8%) for influenza A other than H1N1 virus.The distribution of influenza A/H1N1 cases showed two peaks, one on July (62.9%) and the other one on October (36%), moreover we observed that 155 patients (53%) out of 293 positive for influenza A/H1N1 virus aged under 20 years old. The first positivity peak was found in travelers and the second one, occurred 2-3 months prior to the classic seasonal epidemic influenza, was attributed to autochthonous cases , by which the virus had spread worldwide. The highest proportion of cases were among subjects aged from 0 to 20 years and, over this age the positivity rate decreased proportionally with increasing age, in agreement with data reported in other countries

Presence of carbepenemase-producing Enterobacteriaceae in the River Lambro basin, Italy: might sediment represent an important resistance reservoir?

In the last years, the rapid spread in anthropized ecosystems of pathogens which are resistant to carbapenem antibiotics has raised great concern. In this study, KPC-producing Klebsiella pneumoniae was found in the River Lambro in June 2019, whereas KPC-producing Klebsiella oxytoca and Citrobacter braakii were identified in untreated wastewaters. Susceptibility profiles indicated resistance to imipenem, ertapenem and meropenem. Different carbapenamase genes (blaKPC, blaNDM, blaVIM, blaOXA-48) were also found in the River Lambro, although not associated to living bacteria. The presence of a wide set of carbapenemase genes and resistant pathogens show that river sediments could act as a reservoir of antibiotic resistance potentially threatening human health

Mingling of human and veterinary strains of Staphylococcus aureus : an emerging issue in health-care systems

Aim: Methicillin-resistant Staphylococcus aureus remains a leading cause of hospital and community infections. We report a retrospective molecular characterization of S. aureus strains from different settings: hospital workers and patients, and veterinarian surgeons and pets. Materials and Methods: Eighty-nine S. aureus isolates obtained from nasal swabs of 10 patients, 17 health-care workers (HCWs), 9 pets, and 53 veterinarians were genotypically characterized by means of repetitive extragenic palindromic polymerase chain reaction (Rep PCR) and whole-genome sequencing. Results: Thirteen different sequence types (STs) were detected: ST398, ST22, ST8, ST30, ST15, ST5, ST121, ST45, ST10, ST6, ST34, ST97, and ST1. Two new STs differing from ST22 and ST5 for a single multilocus sequence typing gene were also identified. Rep PCR documented a genetic relationship among isolates obtained from 5 veterinarians and 10 HCWs. Conclusion: The large diversity of S. aureus strains detected may reflect a larger epidemiology within the hospital and community, in which companion animals likely act as a reservoir. We identified the circulation of ST5, ST8, ST15, ST22, ST30, ST45, and ST121 both in the hospital and veterinarian environment. Starting from the idea of a unique setting where our population lives, we consider the relationship between community- and hospital-acquired S. aureus

Imported arboviral infections in Italy, July 2014-October 2015: A National Reference Laboratory report

BACKGROUND: Imported cases of infections due to Dengue (DENV) and Chikungunya (CHIKV) viruses and, more recently, Zika virus (ZIKV) are commonly reported among travelers returning from endemic regions. In areas where potentially competent vectors are present, the risk of autochthonous transmission of these vector-borne pathogens is relatively high. Laboratory surveillance is crucial to rapidly detect imported cases in order to reduce the risk of transmission. This study describes the laboratory activity performed by the National Reference Laboratory for Arboviruses (NRLA) at the Italian National Institute of Health in the period from July 2014 to October 2015. METHODS: Samples from 180 patients visited/hospitalized with a suspected DENV/CHIKV/ZIKV infection were sent to the NRLA from several Italian Hospitals and from Regional Reference Laboratories for Arboviruses, in agreement with the National Plan on human surveillance of vector-borne diseases. Both serological (ELISA IgM test and Plaque Reduction Neutralization Test-PRNT) and molecular assays (Real Time PCR tests, RT-PCR plus nested PCR and sequencing of positive samples) were performed. RESULTS: DENV infection was the most frequently diagnosed (80 confirmed/probable cases), and all four genotypes were detected. However, an increase in imported CHIKV cases (41 confirmed/probable cases) was observed, along with the detection of the first ZIKV cases (4 confirmed cases), as a consequence of the recent spread of both CHIKV and ZIKV in the Americas. CONCLUSIONS: Main diagnostic issues highlighted in our study are sensitivity limitations of molecular tests, and the importance of PRNT to confirm serological results for differential diagnosis of Arboviruses. The continuous evaluation of diagnostic strategy, and the implementation of laboratories networks involved in surveillance activities is essential to ensure correct diagnosis, and to improve the preparedness for a rapid and proper identification of viral threats

In vitro evaluation of antibiotics' combinations for empirical therapy of suspected methicillin resistant Staphylococcus aureus severe respiratory infections

<p>Abstract</p> <p>Background</p> <p>Methicillin resistant <it>Staphylococcus aureus </it>(MRSA) is an increasingly common cause of nosocomial infections, causing severe morbidity and mortality worldwide, and accounting in some hospitals for more than 50% of all <it>S. aureus </it>diseases. Treatment of infections caused by resistant bacterial pathogens mainly relies on two therapeutic modalities: development of new antimicrobials and use of combinations of available antibiotics.</p> <p>Combinations of antibiotics used in the empiric treatment of infections with suspected methicillin resistant <it>Staphylococcus aureus </it>etiology were investigated.</p> <p>Methods</p> <p>Double (vancomycin or teicoplanin with either levofloxacin or cefotaxime) and triple (vancomycin or teicoplanin + levofloxacin + one among amikacin, ceftazidime, cefepime, imipenem, piperacillin/tazobactam) combinations were evaluated by means of checkerboard assay and time kill curves. Mutational rates of single and combined drugs at antimicrobial concentrations equal to the resistance breakpoints were also calculated.</p> <p>Results</p> <p>Vancomycin or teicoplanin + levofloxacin showed synergy in 16/50 and in 9/50 strains respectively, while vancomycin or teicoplanin + cefotaxime resulted synergic for 43/50 and 23/50 strains, respectively. Triple combinations, involving teicoplanin, levofloxacin and ceftazidime or piperacillin/tazobactam gave synergy in 20/25 strains. Teicoplanin + levofloxacin gave synergy in triple combinations more frequently than vancomycin + levofloxacin.</p> <p>For single antibiotics, mutational frequencies ranged between 10^-5and <10^-9for levofloxacin, cefotaxime, amikacin and imipenem, and <10^-9for vancomycin and teicoplanin. When tested in combinations, mutational frequencies fell below 10^-9for all the combinations.</p> <p>Conclusion</p> <p><it>In vitro </it>evidence of synergy between glycopeptides, fluoroquinolones (levofloxacin) and β-lactams and of reduction of mutational frequencies by combinations are suggestive for a potential role in empirical therapy of severe pneumonia with suspected MRSA etiology.</p