Antimicrobial Stewardship in the Covid-19 Pandemic.

During COVID-19 pandemic, implementing and maintaining an antimicrobial stewardship protocol obtained both low rates of MDR microorganisms and low antimicrobial use in an 800-bed hospital network in northern Italy. Infectious diseases specialist consulting was crucial to maintain this protocol active.

Epidemiology of respiratory virus before and during COVID-19 pandemic.

The COVID pandemic has forcefully turned the spotlight on the importance of the diagnosis of respiratory virus infections. Viruses have always been a frequent and common cause of respiratory tract infections. Rapid molecular diagnostics applied to the diagnostics of respiratory virus infections has revolutionized microbiology laboratories only a few years ago. Few studies illustrate the epidemiology of respiratory viruses, and fewer still those that have compared the pre-pandemic to the pandemic period. During the first year of the pandemic (2020-2021) it was clear to everyone to witness a sudden disappearance of the circulation of all the other respiratory viruses, especially those typically isolated during the winter time, such as RSV and Influenza virus. In our study we wanted to verify this phenomenon and to study the epidemiology of our local reality, analyzing three consecutive flu seasons (2018-2019, 2019-2020, 2020-2021). The results lead us to note that the prevalence of positivity to respiratory virus infections went from 49.8% (2018-2019) and 39% (2019-2020) to 13.4% (2020-2021). This decrease is at least partly attributable to the security measures adopted (social distancing and mask), but it certainly opens up new scenarios when the restriction measures will be terminated. We believe such studies can provide real-world evidence of the effectiveness of public health interventions implemented during current and future pandemics.

Molecular diagnosis of SARS-Cov-2, performances and throughput by Direct RT-PCR.

The Covid-19 pandemic has required all laboratories to rapidly and unexpectedly reorganize to cope with the increase in requests for tests in rapid response times and, not least, to provide the shortening of molecular reagents. In order to validate an accurate, faster and cheaper method suitable for large-scale diagnosis of SARS-CoV-2, we evaluated a simplified workflow by Direct RT-PCR on 181 nasopharyngeal swabs on Seegene's automated platform. Direct RT-PCR ensured 99% overall concordance versus standard RNA RT-PCR in samples with Ct values under 35, saving 100% on extraction reagents and providing an approximately three-fold increase in productivity in 24 hours.

Diagnostic performance of an automated chemiluminescence immunoassay for SARS-CoV-2 IgG and IgM antibodies detection: A real life experience.

BACKGROUND: Recently many serological assays for detection of antibodies to SARS-COV-2 virus were introduced on the market. Aim of this study was to assess the diagnostic performance of an automated CLIA for quantitative detection of anti-SARS-CoV-2 IgM and IgG antibodies. METHODS: A total of 354 sera, 89 from consecutive patients diagnosed with COVID-19 (43 mild, 32 severe and 13 critical) and 265 from asymptomatic and negative on rRT-PCR testing healthcare workers, were evaluated for IgM and IgG anti-SARS-CoV-2 antibodies with MAGLUMI immunoassay. RESULTS: The overall sensitivity and specificity were 86.5% (95%CI: 77.6-92.8) and 98.5% (95%CI:96.2-99.6), respectively. PPV, PPN, LR+, LR- and OR were 95.1 (95%CI: 87.8-98.6), 95.6 (95%CI: 92.4-97.7), 57.3 (95%CI: 21.6-152.1), 7.3 (95%CI: 4.31-12.4) and 418.6 (95%CI: 131.2-1335.2), respectively. The levels of SARS-CoV-2 IgM and IgG antibodies were 1.22 â€‹± â€‹1.2 AU/mL and 15.86 â€‹± â€‹24.83 AU/mL, 2.86 â€‹± â€‹2.4 AU/mL and 69.3 â€‹± â€‹55.5 AU/mL, 2.47 â€‹± â€‹1.33 AU/mL and 83.9 â€‹± â€‹83.9 AU/mL in mild, severe and critical COVID-19 groups, respectively. A significant difference in antibody levels between mild and severe/critical subjects has been shown. CONCLUSIONS: The CLIA assay showed good diagnostic performance and a significant association between antibody levels and severity of the disease was found.

Epidemiological and clinical features of imported malaria at the three main hospitals of the Friuli-Venezia Giulia Region, Italy.

BACKGROUND: Imported malaria cases continue to occur in non-endemic regions among travellers returning from tropical and subtropical countries. At particular risk of acquiring malaria is the group of travellers identified as immigrants who return to their home country with the specific intent of visiting friends or relatives (VFRs) and who commonly believe they are immune to malaria and fail to seek pre-travel advice. Our aim was to review the current trends of imported malaria in the three main hospitals of the Friuli-Venezia Giulia region (FVG), North Eastern Italy, focusing in particular on patient characteristics and laboratory findings. METHODS: In this retrospective study, we examined all malaria cases among patients admitted from January 2010 through December 2014 to the emergency department of the three main hospitals located in FVG. RESULTS: During the 5-year study period from 2010 to 2014, there were a total of 140 patients with a diagnosis of suspected malaria and who received microscopic confirmation of malaria. The most common species identified was P. falciparum, in 96 of 140 cases (69%), followed by P. vivax (13%), P. ovale (4%), P. malariae (4%), and mixed infection (4%). The most common reason for travel was VFRs (54%), followed by work (17%), and recent immigration (15%). Moreover, 78% of all patients took no chemoprophylaxis, 80 (79%) of whom were foreigners. Notably, the percentage of Italian travellers who took chemoprophylaxis was only 20% (8 of 39 Italian cases), and the regimen was appropriate in only four cases. Parasitaemia greater than 5% was observed in 11 cases (10%), all due to P. falciparum infection. CONCLUSIONS: We highlight that VFRs have the highest proportion of malaria morbidity and the importance of improving patient management in this category. These data are useful for establishing appropriate malaria prevention measures and recommendations for international travellers.

Evaluation of the new Sysmex UF-5000 fluorescence flow cytometry analyser for ruling out bacterial urinary tract infection and for prediction of Gram negative bacteria in urine cultures.

BACKGROUND: We evaluated the new flow cytometer UF-5000 with a blue semiconductant laser as a screening tool for ruling out urine samples negative for UTI and its ability to predict Gram negatives in culture. METHODS: Flow cytometry and microbiological analysis were performed on 2719 urine samples, sent to our microbiology laboratory with a request for urine culture. RESULTS: UF-5000 showed a very good performance in the screening process. Carryover and cross-contamination was negligible. 797 samples were culture positive at a cut-off of ≥105CFU/mL. ROC curve analysis for BACT count demonstrated AUC between 0.973, on 2714 samples, 0.959, on 1516 female samples, and 0.988 on 1198 male samples, respectively. At the cut-off of BACT ≥58/µL AND/OR YLC ≥150/µL, SE was 99.4%, SP 78.2%, PPV 65.4% and NPV 99.7%; false negatives were 0.6%, avoiding unnecessary cultures in 55.5% of specimens. "Gram Neg?" flag predicted Gram negatives in culture with a SE of 81.6% and SP of 93.3%. CONCLUSION: The new Sysmex UF-5000 showed high diagnostic accuracy in UTI-screening with a very low rate of false negatives. The instrument is capable of predicting Gram negatives with a good SE and a high agreement with the culture, even if this performance needs further evaluation.

Direct molecular detection of pathogens in blood as specific rule-in diagnostic biomarker in patients with presumed sepsis: our experience on a heterogeneous cohort of patients with signs of infective systemic inflammatory response syndrome.

The practical value of blood cultures in the diagnosis of sepsis is impaired by a delay in the turnaround time to result and by the fact that blood culture positive can be found for only about 30% of these patients. Conventional laboratory signs of sepsis and acute phase protein biomarkers are sensitive and easy to use, but often also very nonspecific. Molecular diagnostic reflects currently the most promising avenue to decrease time to result and to influence decision making for antibiotic therapy in the septic host. In this study, we wish to highlight the impact of the LightCycler SeptiFast, a multipathogen probe-based real-time polymerase chain reaction, in the rapid etiological diagnosis of sepsis in patients with clinical and laboratory signs of bloodstream infections. We have evaluated prospectively 830 adult patients with suspected bloodstream infection and at least two criteria of systemic inflammatory response syndrome. In more than 50% of critically ill patients strongly suspected of having sepsis, we arrived to an etiological diagnosis only by the molecular method in a median time of 15 h, with specificity and predictive positive values of 96% and 94%, respectively. We highlight the role of DNAemia as time-critical, high-specificity, etiological, non-culture-based rule-in diagnostic biomarker in patients with presumed sepsis.

When should adenoviral non-gonococcal urethritis be suspected? Two case reports.

The impact of Adenovirus as agent of non-gonococcal urethritis (NGU) is still poorly documented in the literature. We describe two cases showing that adenoviral infection should be reasonably hypothesized in men with dysuria and scant urethral discharge in addition to meatus inflammation and/or edema (meatitis) or conjunctivitis. Case 1: a 55-year-old man came to our observation in July 2012 referring a 5-day-history of intense dysuria and scant mucoid urethral discharge. Physical examination revealed the urethral discharge referred, but also modest meatitis and an intense conjunctival hyperemia on his right eye. Adenoviral infection was investigated and Adenovirus DNA (type 37) was detected in both the urethral and conjunctival swabs. Case 2: a 43-year-old man with intense dysuria, started 4-5 days earlier, came to our attention with his wife in August 2012. Scant urethral mucoid secretions, severe meatal inflammation of the male patient were revealed during physical examination. His wife instead complained of a 2-day history of intense burning eyes. Adenoviral infection was investigated and Adenovirus DNA (type 37) was positive both in the male urethral swab and in his wife's conjunctival swab. Adenovirus seems to cause a distinct and recognisable clinical syndrome in men presenting with urethritis. Studies on the prevalence and role of Adenovirus as a causative agent of urethritis are limited. Moreover, as rapid advanced molecular microbiology is now available, we believe that extending the search to Adenovirus in sexually active men with dysuria, scant discharge in addition to meatitis or conjunctivitis, should be a useful approach improving our understanding about adenoviral NGU, and especially avoiding or stopping unnecessary empirical antibiotic therapy.

Methicillin-resistant Staphylococcus aureus infection rate after implementation of an antibiotic care bundle based on results of rapid molecular screening.

AIM: Colonization with methicillin-resistant Staphylococcus aureus (MRSA) is a risk factor for subsequent invasive MRSA infection, particularly in patients admitted to critical areas. We conducted a surveillance among patients admitted to our Intensive Care Unit (ICU) to determine whether the implementation of a specific MRSA antibiotic care bundle (ACB) based on rapid molecular screening for MRSA and de-colonization, reduced the total MRSA infection rate. MATERIALS AND METHODS: A total of 431 and 577 nasal swabs were obtained from ICU patients at admission from April 2009 through December 2010 (pre-ACB period) and, after the bundle implementation, from January 2011 through December 2012 (post-ACB period), respectively. Nasal swabs were analyzed by the rapid molecular test Xpert MRSA. All patients were followed-up during their whole ICU stay to determine whether they developed MRSA infection. RESULTS: Overall, 31 out of 431 (7.1%) patients were colonized with MRSA at admission during the pre-ACB period and 49 out of 577 (8.4%) were colonized with MRSA during the post-ACB period. The rate of MRSA infection in ICU significantly declined from 2% in pre-ACB to 0.3% in post-ACB, with a total decrease of 100% in two consecutive semesters between July 2011 and July 2012 (p<0.001). CONCLUSION: The analysis demonstrated a significant decline in MRSA infections following the introduction of active rapid molecular surveillance and the specific ACB at our ICU and in the risk associated with MRSA bacteremia.

An antibiotic care bundle approach based on results of rapid molecular screening for nasal carriage of methicillin-resistant Staphylococcus aureus in the intensive care unit.

The potential role of active methicillin-resistant Staphylococcus aureus (MRSA) surveillance in the intensive care unit (ICU), has been recently proposed as a guide for antibiotic treatment in patients suspected of being infected with MRSA by using an antibiotic care bundle (ACB) approach. A group of 376 consecutive ICU patients were prospectively screened for nasal carriage of MRSA using a real-time polymerase chain reaction test. The study group consisted of 244 (64.9%) males and (35.1%) females, with a median age of 64 (range 17-95 years). Overall, 26 (6.9%) patients were positive for MRSA, while 350 (93.1%) were MRSA-negative. No difference was observed in gender and age between groups. During ICU stay, 9 (2.4%) patients developed generalized MRSA infection, of whom 8 out of 26 (30.8%) were MRSA-carriers and one out of the 350 (0.3%) was MRSA-negative. Thus, a strong relationship between MRSA infection and MRSA carriage (relative risk=107.7, 95% confidence interval=14.0-828.5, p<0.0001) was found. Subsequently, in our ICU, we developed and introduced a new ACB approach based on rapid nasal screening results for improving the management of critically ill patients. The use of anti-MRSA agents should be re-evaluated daily on the basis of clinical and laboratory features, with positive cultures from sterile site or signs of active infection supporting prolongation of empirical treatment. On the contrary, MRSA-negative clinical cultures support a de-escalation strategy. In conclusion, the early identification of MRSA-carriers using a rapid molecular screening is safe and accurate, allowing MRSA-positive patients, who will more likely develop MRSA infections, to be detected.

Evaluation of the Sysmex UF1000i flow cytometer for ruling out bacterial urinary tract infection.

BACKGROUND: Urine culture is one of the most frequently requested tests in microbiology, and it represents the gold standard for the diagnosis of UTIs. Considering the high prevalence of negative results and the long TAT of the culture test, the use of a rapid and reliable screening method is becoming more and more important, as it reduces the workload, the TAT of negative results, and above all, unnecessary antibiotic prescription. METHODS: The Sysmex UF1000i is a new urine flow cytometry analyzer capable of quantifying urinary particles, including BACT, WBCs, and YLCs. To evaluate the analytical performance of the UF1000i as a method for ruling out UTIs, we examined 1349 urine samples and compared the UF1000i results with standard urine culture results. RESULTS: With instrument cut-off values of 170BACTx10(6)/L and 150WBCsx10(6)/L, we obtained a sensitivity of 98.8%, a specificity of 76.5%, a NPV of 99.5%, and four false negative results (1.2%), avoiding the culture of 57.1% of samples. CONCLUSION: The Sysmex UF1000i was capable of improving the efficiency of a routine microbiology laboratory by processing 100samples/h and providing negative results in a few minutes, thus reducing unnecessary testing with an acceptable number of false negative results. In addition, the preliminary evaluation of B_FSC and B_FLH parameters from bacteria histograms seems to be useful for the distinction of bacterial strains detected (Gram-negatives versus Gram-positives). In fact when B_FSC was less than 30 ch, it allowed the distinction of Gram-negative bacteria in 97% of the samples.

Molecular identification of bloodstream pathogens in patients presenting to the emergency department with suspected sepsis.

The rapid detection of pathogens in blood is critical for a favorable outcome of patients with suspected sepsis. Although blood culture (BC) is considered the criterion standard for diagnosis of bloodstream infection, it often takes several days to detect the causative organism. In this study, we compared BC with a commercially available multiplex real-time polymerase chain reaction (PCR) assay to detect bacteria and fungi in blood samples from 144 patients admitted to the emergency department with suspected sepsis. Of 144 blood samples examined, 91 (63%) were negative by both methods and 53 (37%) were positive by at least one of the two methods. In 30 among all positive cases (56.6%),both methods identified the same organisms, in 13 cases (24.5%), BC identified organisms not detected by real-time PCR,and in 10 cases (18.9%), SeptiFast PCR assay gave positive results, whereas the BC was negative. In this study, we wished to compare SeptiFast results obtained by standard procedures, but future clinical studies are necessary to define SeptiFast PCR as support for BC in the early diagnosis of severe bloodstream infections.

Aeromonas hydrophila ecthyma gangrenosum without bacteraemia in a diabetic man: the first case report in Italy.

Ecthyma gangrenosum is a well recognized cutaneous manifestation of severe, invasive infection by Pseudomonas aeruginosa usually in immunocompromised and critically ill patients. This type of infection is usually fatal. Aeromonas infection is infrequently reported as the cause of ecthyma gangrenosum. Here we show the first case described in Italy of Aeromonas hydrophila ecthyma gangrenosum in the lower extremities in an immunocompetent diabetic without bacteraemia. A 63-year-old obese diabetic male was admitted with an ulcer on his left leg, oedema, pain and fever. Throughout his hospitalization blood cultures remained sterile, but a culture of A. hydrophila was isolated following punctures from typical leg pseudomonal-ecthyma gangrenosum lesions developed after admission. The patient, questioned again, stated that a few days before he had worked in a well near his house without taking precautions. We conclude that early diagnosis and suitable antibiotic therapy are important for the management of ecthyma gangrenosum. The typical presentation of soft tissue infection of A. hydrophila should mimic a Gram-positive infection, which may result in a delay in administration of appropriate antibiotics. Moreover, A. hydrophila should be considered a possible agent for non-pseudomonal ecthyma gangrenosum in a diabetic man with negative blood cultures, in presence of anamnestical risk factors.

Direct antifungal susceptibility testing of positive Candida blood cultures by sensititre YeastOne.

Invasive candidiasis is associated with high morbidity and mortality. Differences in the virulence and susceptibility of the various Candida spp. to antifungal drugs make the identification and rapid MIC determination very important for clinical management. The aim of this study was to improve the turnaround time (TAT) for antimicrobial test generation by susceptibility testing directly from the bottle of blood culture positive for yeasts, circumventing the isolation process and thereby generating an accurate antifungal MIC determination as quickly as possible. Sensititre YeastOne was used by direct inoculation from positive blood culture bottles in 40 cases of candidaemia. All the results were compared with those obtained using standard laboratory procedures after subculturing from a positive bottle onto solid media. The results obtained from direct inoculation of Sensititre YeastOne compared with tests carried out using standard procedures show that out of a total of 40 strains tested no very major errors or major errors and only 4 minor errors occurred (98% agreement rate out of a total of 240 drug/bug combinations tested), thus generating an accurate antifungal MIC determination and saving an average time of 24 hours compared with the time required for the standard procedures traditionally used.

[Disappearance of Streptococcus pyogenes macrolide resistance in an area of northeastern Italy: a possible link with rational long-acting macrolide consumption]. / Scomparsa della resistenza di Streptococcus pyogenes ai macrolidi in un'area del nord est e possibile nesso con il razionale utilizzo di molecole long-acting.

Many studies have shown a correlation between higher consumption of long-acting macrolides and the development of resistance of S. pyogenes but, to our knowledge, no studies have reported the disappearance of S. pyogenes macrolide resistance. We evaluated the possible relationship between the rational use of long-acting macrolide consumption and the disappearance of S. pyogenes erythromycin resistance in an area of northeastern Italy, the district of Pordenone (approximately 300,000 inhabitants). The emerging use of new long-acting macrolides, especially since 1993, has caused a great increase in total macrolide consumption (expressed as defined daily doses per 1,000 inhabitants per day; DDDs), followed by a steady increase in the percentage of S. pyogenes resistant to erythromycin (from 4% in 1994 to 56.3% in 2000). Subsequently, from 2000 to 2007, the maintenance of steady-high DDDs of clarithromycin but low DDDs of azithromycin resulted in a sharp decrease in the percentage of S. pyogenes resistance to erythromycin (from 33.3% in 2001 to 0.2% in 2008). Disappearance of S. pyogenes erythromycin resistance in the last few years, compared with data of long-acting macrolide consumption from 2000 to 2007, suggests that S. pyogenes resistance to erythromycin is more likely associated with the specific type of compound used rather than with total consumption of long-acting macrolides.

Improving the efficiency and efficacy of pre-analytical and analytical work-flow of urine cultures with urinary flow cytometry.

Quantitative urine culture is one of the most frequently requested tests in microbiology laboratories. An automated system for screening purposes is strongly needed to save technical staff time and obtain rapid results. Our study investigated 1.047 urine samples collected from inpatients and outpatients with a commercial vacutainer system (Becton Dickinson, Milan, Italy) and compared a second-generation flow cytometry (Sysmex UF100, Dasit, Cornaredo, Italy) with standard urine culture tested on agar plated by means of 10 microliter loop. The specimens were screened and cultured on receipt. The results obtained with Sysmex UF-100 are very interesting, especially if this analyzer is used as a screening method for negative urine samples, and comparable to data obtained from culture examination. In fact, considering together bacteria and leukocyte count (> 4500 bacteria and/or > 50 leukocytes/microL) we obtained a negative predictive value of 99.5% in comparison with the standard culture method. The classical culture method needs 24 hours for a result, whereas the Sysmex UF-100 analyzer gives results in a few minutes, thus reducing the microbiology turn around time (TAT) with obvious benefits for patients and physicians.