Evaluation of pregenomic HBV RNA in HBeAg-negative patients.

The distinction between chronic HBeAg-negative hepatitis (CHB) and chronic HBeAg-negative infection (CIB) can be challenging and important for providing advice on prognosis, as well as determining need for treatment. The aim of the present study was to evaluate pgRNA levels in treatment-naïve HBeAg-negative chronic HBV-infected patients. In addition, pgRNA levels were compared to traditional markers in order to assess their clinical utility. A retrospective study was carried out, including 85 cases of CHBs and 74 CIBs. Globally, when the virological markers (pgRNA, qHBsAg, and HBV DNA) were analyzed, significant differences were found between the CHB and CIB groups (P<0.001). Overall, positive correlations were demonstrated, as follows: between pgRNA levels and qHBsAg (Spearman r=0.30, P<0.001), between pgRNA and HBV DNA (Spearman r=0.73, P<0.001), and between pgRNA and ALT (Spearman r=0.67, P<0.001). Out of the 85 CHB patients, 82 (96.5%) agreed to start treatment. At baseline, 38/82 patients, as well as the 3 untreated CHB patients, had undetectable pgRNA levels. The 74 CIB carriers also had undetectable pgRNA levels. During the follow-up period, no patients experienced viral reactivation or progression of liver disease. These results suggest that the addition of plasmatic HBV-pgRNA levels to the traditional diagnostic flowchart of HBeAg-negative patients may improve the correct identification of cases at risk, especially patients with occasional increases in HBV viremia.

Epidemiology of Invasive Pulmonary Aspergillosis Among Intubated Patients With COVID-19: A Prospective Study.

BACKGROUND: We evaluated the incidence of invasive pulmonary aspergillosis among intubated patients with critical COVID-19 and evaluated different case definitions of invasive aspergillosis. METHODS: Prospective, multicenter study in adult patients with microbiologically confirmed COVID-19 receiving mechanical ventilation. All included participants underwent a screening protocol for invasive pulmonary aspergillosis with bronchoalveolar lavage galactomannan and cultures performed on admission at 7 days and in case of clinical deterioration. Cases were classified as coronavirus-associated pulmonary aspergillosis (CAPA) according to previous consensus definitions. The new definition was compared with putative invasive pulmonary aspergillosis (PIPA). RESULTS: 108 patients were enrolled. Probable CAPA was diagnosed in 30 (27.7%) patients after a median of 4 (2-8) days from intensive care unit (ICU) admission. Kaplan-Meier curves showed a significantly higher 30-day mortality rate from ICU admission among patients with either CAPA (44% vs 19%, P = .002) or PIPA (74% vs 26%, P < .001) when compared with patients not fulfilling criteria for aspergillosis. The association between CAPA (OR, 3.53; 95% CI, 1.29-9.67; P = .014) or PIPA (OR, 11.60; 95% CI, 3.24-41.29; P < .001) with 30-day mortality from ICU admission was confirmed, even after adjustment for confounders with a logistic regression model. Among patients with CAPA receiving voriconazole treatment (13 patients; 43%) a trend toward lower mortality (46% vs 59%; P = .30) and reduction in galactomannan index in consecutive samples were observed. CONCLUSIONS: We found a high incidence of CAPA among critically ill COVID-19 patients and its occurrence seems to change the natural course of disease.

Genetic and Functional Characterization of Toll-Like Receptor Responses in Immunocompetent Patients With CMV Mononucleosis.

Background: Human cytomegalovirus (CMV) modulates both innate and adaptive immune responses. However, limited data are available on the role of receptors of innate immunity, such as toll-like receptors (TLRs) in contributing to antiviral responses and inflammation. Objectives: The aim of this translational study was to characterize TLR responses in immunocompetent patients with primary and symptomatic CMV infection. Study Design: The study population consisted of 40 patients suffering from CMV mononucleosis and 124 blood donors included as controls. We evaluated the association between TLR2, 3, 4, 7 and 9 gene single nucleotide polymorphism (SNP) and susceptibility to symptomatic CMV infection in immunocompetent adults. Additionally, functional TLR-mediated cytokine responses in supernatants of short-term cultures of whole blood from patients with CMV mononucleosis and blood donors were evaluated. Results: TLR2 and TLR7/8 responses were altered in CMV infected patients as compared to healthy donors and were associated with the release of higher levels of the pro-inflammatory cytokines IL-6 and TNF-α, but not of the anti-inflammatory mediator IL-10. The analysis on the TLR SNPs indicated no difference between patients with CMV infection and the control group. Conclusions: No variation in the TLR2,3,4,7 and 9 genes was associated to the development of symptomatic CMV infection in immunocompetent adults. Nevertheless, TLR-mediated responses in CMV-infected patients appeared to be skewed toward a pro-inflammatory profile, which may contribute to the development of inflammatory symptoms during the CMV mononucleotic syndrome.

Recent Advances in the Evaluation of Serological Assays for the Diagnosis of SARS-CoV-2 Infection and COVID-19.

Introduction: Few data on the diagnostic performance of serological tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are currently available. We evaluated sensitivity and specificity of five different widely used commercial serological assays for the detection of SARS-CoV-2-specific IgG, IgM, and IgA antibodies using reverse transcriptase-PCR assay in nasopharyngeal swab as reference standard test. Methods: A total of 337 plasma samples collected in the period April-June 2020 from SARS-CoV-2 RT-PCR positive (n = 207) and negative (n = 130) subjects were investigated by one point-of-care lateral flow immunochromatographic assay (LFIA IgG and IgM, Technogenetics) and four fully automated assays: two chemiluminescence immunoassays (CLIA-iFlash IgG and IgM, Shenzhen YHLO Biotech and CLIA-LIAISON® XL IgG, DiaSorin), one electrochemiluminescence immunoassay (ECLIA-Elecsys® total predominant IgG, Roche), and one enzyme-linked immunosorbent assay (ELISA IgA, Euroimmune). Results: The overall sensitivity of all IgG serological assays was >80% and the specificity was >97%. The sensitivity of IgG assays was lower within 2 weeks from the onset of symptoms ranging from 70.8 to 80%. The LFIA and CLIA-iFlash IgM showed an overall low sensitivity of 47.6 and 54.6%, while the specificity was 98.5 and 96.2%, respectively. The ELISA IgA yielded a sensitivity of 84.3% and specificity of 81.7%. However, the ELISA IgA result was indeterminate in 11.7% of cases. Conclusions: IgG serological assays seem to be a reliable tool for the retrospective diagnosis of SARS-CoV-2 infection. IgM assays seem to have a low sensitivity and IgA assay is limited by a substantial rate of indeterminate results.

Role of cultural analysis in patients with indwelling ureteral stent submitted to ureteroscopy for stones.

BACKGROUND: Aim of our study is to analyze the incidence of postoperative infectious complications and to assess its predictors in patients with indwelling ureteral stent treated with ureteroscopy (URS). METHODS: We retrospectively evaluated data of patients treated with URS from January 2017 to July 2018 at our center. We included 88 consecutive patients with available stent culture (SC) and urine culture (UC). Cefoxitin 2 g IV was given as prophylaxis in all patients with negative preoperative UC; otherwise, the choice of antibiotic was based on antibiogram. Ureteral stent was removed before URS procedure and analyzed. No postoperative antibiotic was given. Multivariable logistic regression analysis was built to assess preoperative predictors of postoperative infectious complications. RESULTS: Nineteen patients (22%) developed postoperative infectious complications and fever was the most common one. E. faecalis, which is not responsive to common prophylaxis schemes in force in our institution, was the most frequent pathogen isolated. Overall, 26% of patients were found to have a discordance between SC and UC. At multivariable logistic regression analysis preoperative SC positivity (Odds Ratio [OR]: 11.00, 95% Confidence Interval [CI]:1.08-111.41, P=0.04) was the only significant predictor of postoperative infectious complications. CONCLUSIONS: About one to five patients treated with URS developed an infectious complication and E. faecalis and E. coli were the most frequent pathogen isolated. A positive SC is the only independent risk factor for postoperative infection: consequently, an early SC analysis could allow a prompt antibiotic therapy in all patients with positive SC even if mildly symptomatic.

Syphilis in a high-density urban area in the North of Italy.

Although far less common now than in the past, syphilis continues to pose a danger to public health and should not be overlooked. In this study, we evaluated the presence and characteristics of syphilis in a group of patients attending an STI Clinic in the North of Italy. A retrospective study was carried out, analysing the data from the 5609 subjects who attended the STI Clinic of St. Orsola-Malpighi Hospital (Bologna) for syphilis screening from January 2016 to December 2017. Globally, 692 patients (12.3%) were found positive for treponemal tests, with a significant difference between males and females (16.6% vs 4.1%; P<0.0001). Moreover, positive women were more likely foreign (63.3%), in contrast to men, who were more likely Italian (86.1%; P<0.0001). A total of 306 patients (44.2%), mainly males (47% vs 25%; P=0.0003), received a diagnosis of early syphilis. These cases peaked among patients 35-44 years (31%) and 25-34 years (26.8%). Overall, 32.9% of the women found positive for treponemal tests were pregnant. Among them, 84.6% were foreign (mainly from Eastern Europe) and 38.4% received a diagnosis of early syphilis. No cases of mother-to-child syphilis were found. The presence of an HIV-syphilis co-infection was found in 21.5% of patients with early syphilis, with a significant association with the male sex (P<0.009). In-depth knowledge of the characteristics of syphilis could help set up effective strategies for its control.

Prevalence and antimicrobial resistance of genital Mollicutes in Italy over a two-year period.

Knowledge of the prevalence and antimicrobial susceptibility of genital Mollicutes is crucial to offer guidelines for empirical treatments. The aim of this study was to investigate the prevalence and the resistance profile of Mycoplasma hominis (MH) and Ureaplasma urealyticum/Ureaplasma parvum (UU/UP) in genital samples over a two-year period in Bologna, Italy. From January 2015 to December 2016, data on all the subjects providing uro-genital specimens for Mollicutes detection by culture were analyzed. A total of 4660 subjects (84.4% females) were enrolled and an overall Mollicutes prevalence of 30.9% was found. Women turned positive for Mollicutes infection twice as often as men (33.3% vs 17.8%) and the detection rate progressively decreased with increasing age. Ureaplasmas represented the commonest species isolated (overall prevalence: 24.2%), whereas mixed infections (6.5%) and MH single infections (3.9%) were far less common. Ureaplasma species showed significant levels of quinolone resistance, especially to ciprofloxacin (77%), whereas MH strains were non-susceptible to azithromycin and roxithromycin in about 90% of cases. Mollicutes co-infections showed a more severe resistance pattern than single infections. Over time, the resistance rate for azithromycin and roxithromycin increased significantly. Globally, our results revealed that minocycline and doxycycline can still be first-line drugs for Mollicutes treatment.

Emergence of Carbapenem-Resistant Klebsiella pneumoniae: Progressive Spread and Four-Year Period of Observation in a Cardiac Surgery Division.

Frequent use of carbapenems has contributed to the increase to K. pneumoniae strains resistant to this class of antibiotics (CRKP), causing a problem in the clinical treatment of patients. This investigation reports the epidemiology, genetic diversity, and clinical implication of the resistance to drugs mediated by CRKP in our hospital. A total of 280 K. pneumoniae strains were collected; in particular 98/280 (35%) were CRKP. Sequencing analysis of CRKP isolated strains showed that 9/98 of MBL-producing strains carried the bla VIM-1 gene and 89/98 of the isolates were positive for bla KPC-2. Antimicrobial susceptibility tests revealed a complete resistance to third-generation cephalosporins and a moderate resistance to tigecycline, gentamicin, and fluoroquinolones with percentages of resistance of 61%, 64%, and 98%, respectively. A resistance of 31% was shown towards trimethoprim-sulfamethoxazole. Colistin was the most active agent against CRKP with 99% of susceptibility. Clonality was evaluated by PFGE and MLST: MLST showed the same clonal type, ST258, while PFGE analysis indicated the presence of a major clone, namely, pulsotype A. This finding indicates that the prevalent resistant isolates were genetically related, suggesting that the spread of these genes could be due to clonal dissemination as well as to genetic exchange between different clones.

Toxoplasmosis in pregnancy in an area with low seroprevalence: is prenatal screening still worthwhile?

BACKGROUND: The effectiveness of Toxoplasma gondii (Tg) screening during pregnancy in areas with a low prevalence of the infection is debated. We investigate the Tg serological status, the rate of primary infection in a cohort of pregnant women and the rate of congenital toxoplasmosis among their infants during a 3-year period in an urban area with low Tg prevalence. METHODS: Demographic and Tg serological data for all pregnant women delivering from January 2009 to December 2011 were collected. All pregnant women with primary Tg infection during pregnancy and their infants were included in the study. RESULTS: In early pregnancy, 10,347 women underwent prenatal screening and 2308 (22.3%) had anti-Tg. The seroprevalence among non-native women was significantly higher than that among native women [32.8% vs. 19.1%, relative risk: 1.71, P < 0.001]. The incidence rate of primary Tg infection during pregnancy was 0.77%. Immigrant women were more likely to be infected during pregnancy than Italian women (relative risk: 4.88, P < 0.001). Tg infection was more frequent in women coming from Africa, Asia, Eastern Europe and South America. The CT incidence rate was 0.06%. All congenitally infected infants were born to immigrant mothers. CONCLUSIONS: Tg infection during pregnancy and congenital disease are more frequent in non-native mothers and their infants. Measures to prevent Tg exposition must be carefully explained to pregnant women, with a focus on specific habits in non-native women. Prenatal screening is still effective to select women for prenatal therapy aiming to decrease vertical transmission and to identify foetuses/newborns with congenital disease that could benefit from pre/postnatal antiparasitic therapy.

Evaluation of the BioPlex 2200 syphilis system as a first-line method of reverse-sequence screening for syphilis diagnosis.

Despite recent technological advances, the diagnosis of syphilis remains a challenging enterprise. Actually, most high-volume laboratories have adopted the "reverse algorithm" due several factors, including the potential to automate testing. Recently, immunoassays processed on random-access systems have been proposed as screening tests. The purpose of this study was to evaluate diagnostic performances of BioPlex 2200 Syphilis IgG and BioPlex 2200 Syphilis IgM, tests based on Multiplex Flow technology, in comparison with the performance of Architect Syphilis TP, a chemiluminescent immunoassay for the detection of IgG and/or IgM anti-Treponema pallidum antibodies. A retrospective study was performed with a panel of 100 blood donor sera, a panel of 350 clinical and laboratory-characterized syphilitic sera, and 170 samples obtained from subjects with potentially interfering conditions. Moreover, 200 unselected samples submitted to the Microbiology Laboratory of St. Orsola Hospital in Bologna for routine screening for syphilis were evaluated. As confirmatory tests, T. pallidum hemagglutination and Western blot assays were used. Considering the IgG Western blot (WB) assay to be the gold standard method, BioPlex 2200 Syphilis IgG specificity was far higher than Architect Syphilis TP specificity (89.7% versus 78.4%, respectively), whereas the sensitivity was 100% for both automated methods. Compared to the IgM WB assay, BioPlex 2200 Syphilis IgM performed with a specificity of 94.9%, whereas the sensitivity was 84.8%. Considering the excellent ease of use and automation, the high sample throughput and its valuable analytical performances, BioPlex Syphilis 2200 IgG could represent a suitable choice for high-volume laboratories. BioPlex Syphilis 2200 IgM could be considered a good addition to IgG testing for uncovering active infections.

[ProACT (Adjustable Continence Therapy) implants in case of failure of other techniques for urinary incontinence]. / Reimpianto del sistema ProACT in caso di fallimento precoce nella cura dell'incontinenza urinaria dopo prostatectomia radicale.

BACKGROUND: Urinary incontinence after radical prostatectomy is one of the most feared problems. It can affect almost 40% of patients, with different degrees of severity according to each specific case. The aim of this work is to analyze our experience in ProACT (Adjustable Continence Therapy) implants, especially in case of failure of other techniques. METHODS: Between November 2007 and December 2010, 31 patients with post-radical prostatectomy incontinence underwent a ProACT implant. Eight patients had their device explanted (in local anesthesia): in two cases the device spontaneously broke, three of them migrated in the urethra (one patient received radiation therapy), another one was infected in the device site (one in BCG treatment for non-muscle invasive bladder cancer), two devices were wrongly placed. Seven of these patients had had their device replaced with success. Using pad score, incontinence was classified as mild, moderate and severe. Overall, the total amount of procedures, most of them fluoroscopic-guided in spinal anesthesia, were 38; the average duration of the surgery was 37.6 minutes. In one patient with impaired balloon volume due to monolateral device malfunction, we noticed good results in controlling incontinence; therefore, we successfully applied the same technique in other four cases with previous partial results. RESULTS: With a total amount of 28 implants, we had 17 (60.7%) complete responses, 6 (25%) partial and 4 (14.3%) failures. We had 4 post-radiotherapy implants: one was completely dry, two were in balloon adjustment, and one of them had a replacement due to urethral erosion of the first implant.
All patients with impaired balloon inflation were satisfied: one was completely dry and three had sensible improvement. CONCLUSIONS: The ProACT is a minimally invasive surgical therapy for post-radical prostatectomy urinary incontinence. Early failure is frequent and is mainly due to rupture and migration of the device. In these cases the solution can be the replacement, even with impaired balloon inflation.

Staphylococcus aureus nosocomial infections: the role of a rapid and low-cost characterization for the establishment of a surveillance system.

Continuous surveillance on resistance patterns and characterization of Staphylococcus aureus represent simple and low-cost techniques to understand and evaluate the effectiveness of infection control and antimicrobial prescribing measures. In this study we analyzed the antibiotic susceptibility and trends for S. aureus strains collected from bacteraemia cases in a five year period. Between 2004 and 2008 we noted a progressive decrease in the number of S. aureus isolates compared to all pathogens from clinical specimens and S. aureus bloodstream infections (BSI) reflected a similar trend. In particular we analyzed 185 isolates from blood cultures: 89 isolates were MSSA and 96 isolates were MRSA. Molecular SCCmec typing of these strains showed an absolute prevalence of types I and II, whereas five spa types from 96 isolates were obtained. Resistance pattern analysis allowed us to place MRSA strains into 12 antibiotypes and the major antibiotype was resistant to penicillin, gentamicin, erythromycin, clindamycin and ciprofloxacin. The predominant antibiotype among the MSSA isolates was resistant only to penicillin. In addition, 19.1% of MSSA are susceptible to all antibiotics tested. We also found a close association between antibiotyping 1 and genotyping t002/SCCmecI of MRSA strains, suggesting a nosocomial scenario dominated by a few particular clones.

In vitro activities of several antimicrobial agents against recently isolated and genotyped Chlamydia trachomatis urogenital serovars D through K.

A systematic evaluation of the susceptibility of all Chlamydia trachomatis urogenital serovars (D through K) to levofloxacin, erythromycin, doxycycline, clarithromycin, and azithromycin was performed. All C. trachomatis serovars had comparable susceptibilities with respect to the various antimicrobials tested, thus confirming the homogeneous data so far obtained regarding the susceptibility of C. trachomatis to antimicrobial agents.

Tissue engineering in urinary bladder: morphological and functional characterization.

INTRODUCTION: In the last years, tissue engineering has attracted lots of researchers, in urology too. This is due to the possibility to use this technique in several pathologies' therapies, which generally require reconstructive surgical solutions. Our work's aim is to evaluate morphological and functional aspects of cultivated urothelial and detrusorial tissues, both in "monolayer growth" and on scaffolds, in order to understand the chance of using them in reconstructive surgery. MATERIALS AND METHODS: Tissue cultures of detrusorial and urothelial cells have been obtained from animals (pigs, rabbits) and men. The urothelial nature of obtained cells has been demonstrated by traditional histological observation (Hematoxylin - Eosin), by immuno-fluorescence assay (specific for cyto-keratins antibodies), by immuno-histo-chemistry techniques (using specific cyto-keratins 7, 17, and 20 antibodies). Detrusorial tissue has been studied by using antibodies specific for alpha-actin. RESULTS: Urothelial and smooth muscle cells, when isolated and expanded in vitro, keep the typical characteristics of original tissue, as showed by classical histological observation (H-E), immuno-histo-chemistry and immuno-fluorescence assays. These results were positive both in monolayer colonies and on scaffolds. In vitro results are encouraging and they demonstrate that it is possible to obtain in vitro vesical tissue that could have analogous characteristics to the original organ; even though clinical utilisation of this technique must be more investigated, both in vitro and in vivo.

Laboratory diagnosis of syphilis with automated immunoassays.

The serological detection of specific antibodies to Treponema pallidum is of particular importance in the diagnosis of syphilis. The purpose of this study was to evaluate diagnostic performances of automated immunoassays in comparison with T. pallidum hemagglutination test (TPHA) and Western Blot (WB). The retrospective study was performed with different panels of sera: 244 clinical and serological characterized syphilitic sera and 203 potentially interfering samples. All the sera were tested by Enzygnost Syphilis, ARCHITECT Syphilis TP, TPHA, and homemade WB. The diagnostic performances of the two assays were very similar: both Enzygnost Syphilis and ARCHITECT Syphilis TP performed with a sensitivity of 99.2%, whereas the specificity was 98.5 and 98.4%, respectively. Considering the suitability for automation, both immunoassays may represent a good choice as a screening test. However, the use of a confirmatory test, such as TPHA or WB, remains a must in order to avoid false-positive results.

Sensitivity of Chlamydia suis to cathelicidin peptides.

Nine Chlamydia suis isolates, obtained from pigs with conjunctivitis, were molecularly characterized by ompA sequencing and their in vitro susceptibility to six cathelicidin peptides (SMAP-29, BAC-7, BMAP-27, BMAP-27, BMAP-28, PG-1, LL-37) determined in cell culture. SMAP-29 was the most active peptide, reducing the intracellular inclusion number by > or =50% at a concentration of 10 microg/ml (3 microM) in six of the nine isolates tested. Three molecularly identical isolates were insensitive at a concentration as high as 80 microg/ml (25 microM). Of the remaining cathelicidin peptides tested, BAC-7 and BMAP-27 were active against six C. suis isolates at a concentration of 80 microg/ml (25 and 26 microM, respectively). Cathelicidins LL-37 and PG-1 did not show any anti-chlamydial activity at 80 microg/ml.

A decrease in the immunoglobulin G antibody response against the VlsE protein of Borrelia burgdorferi sensu lato correlates with the resolution of clinical signs in antibiotic-treated patients with early Lyme disease.

The purpose of this study was to evaluate the diagnostic performance of the LIAISON Borrelia Screen (Diasorin, Saluggia, Italy), a new automated immunoassay based on the chemiluminescent technology (chemiluminescence immunoassay). To assess whether a decrease in a negative value in the anti-VlsE immunoglobulin G (IgG) antibody titer was correlated with a positive response to treatment, a group of serially collected serum samples from 67 patients with culture-confirmed erythema migrans was retrospectively studied. All the patients had been treated with antibiotics and were free of disease within 3 to 6 months of follow-up. All the 15 patients who were found to be IgG positive at the time of enrollment and who were bled at least four times during the follow-up became IgG seronegative at 2 to 6 months posttreatment. These results indicate that a decline in the anti-VlsE antibody titer coincides with effective antimicrobial therapy in patients with early localized Lyme disease.

Evaluation of LIAISON Treponema Screen, a novel recombinant antigen-based chemiluminescence immunoassay for laboratory diagnosis of syphilis.

The purpose of this study was to evaluate the diagnostic performance of LIAISON Treponema Screen (DiaSorin, Saluggia, Italy), a new automated chemiluminescence immunoassay (CLIA), in comparison with that of rapid plasma reagin (RPR) and the following currently used treponemal tests: hemagglutination test (TPHA), immunoenzymatic assay (EIA), and Western blot (WB). First, a retrospective study was performed with a panel of 2,494 blood donor sera, a panel of 131 clinical and serologically characterized syphilitic sera, and 96 samples obtained from subjects with potentially interfering diseases or conditions. A prospective study was also performed by testing 1,800 unselected samples submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna, Italy, for routine screening for syphilis. As expected, RPR was the least specific method, especially when potentially cross-reacting sera were tested. On the contrary, all of the treponemal tests proved to be very specific (99.9%) and they performed with the following sensitivities: 100% (WB), 99.2% (CLIA), 95.4% (EIA), and 94.7% (TPHA).

Activity of cathelicidin peptides against Chlamydia spp.

The in vitro activity of six cathelicidin peptides against 25 strains of Chlamydia was investigated. SMAP-29 proved to be the most active peptide, reducing the inclusion numbers of all 10 strains of Chlamydia trachomatis tested by > or =50% at 10 microg/ml. This peptide was also active against C. pneumoniae and C. felis.