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Environmental contamination by SARS-CoV-2 from patients with COVID-19 undergoing noninvasive ventilation (NIV) in the ICU is still under investigation. This study set out to investigate the presence of SARS-CoV-2 on surfaces near subjects receiving NIV in the ICU under controlled conditions (ie, use of dual-limb circuits, filters, adequate room ventilation).This was a single-center, prospective, observational study in the ICU of a tertiary teaching hospital. Four surface sampling areas, at increasing distance from subject's face, were identified; and each one was sampled at fixed intervals: 6, 12, and 24 h. The presence of SARS-CoV-2 was detected with real-time reverse transcriptase-polymerase-chain-reaction (RT-PCR) test on environmental swabs; the RT-PCR assay targeted the SARS-CoV-2 virus nucleocapsid N1 and N2 genes and the human RNase P gene as internal control.In a total of 256 collected samples, none were positive for SARS-CoV-2 genetic material, whereas 21 samples (8.2%) tested positive for RNase P, thus demonstrating the presence of genetic material unrelated to SARS-CoV-2.Our data show that application of NIV in an appropriate environment and with correct precautions leads to no sign of surface environmental contamination. Accordingly, our data support the idea that use of NIV in the ICU is safe both for health care workers and for other patients.

A significant weight gain has been reported in HIV-infected patients starting combination antiretroviral therapy (cART) including integrase strand transfer inhibitors, but clinical data about changes in body fat mass are still lacking. An observational retrospective analysis was made to evaluate changes in body fat mass and weight in 39 cART-naive patients initiating a first antiretroviral treatment, including tenofovir disoproxil fumarate/emtricitabine plus raltegravir (RAL) or darunavir/ritonavir (DRV/r), and who had a follow-up of at least 12 months and a whole-body dual-energy X-ray absorptiometry performed at baseline and after 12 months. After 12 months, changes in weight and total fat mass were comparable and statistically not significant in both groups. The median increase [interquartile range (IQR)] in weight was +2.02 kg (+1.19, +2.95; p = .378) in RAL group, and +1.71 kg (+0.89, +2.54; p = .449) in DRV/r group. The median increase in body fat mass (IQR) was +1.27 kg (+1.09, +1.43; p = .278) in RAL group, and +1.04 kg (+0.89, +1.22; p = .781) in DRV/r group. In conclusion, in our study, an initial regimen including RAL plus tenofovir/emtricitabine after 12 months led to a small and nonsignificant increase in weight and body fat mass, and changes were comparable with a DRV/r-based initial regimen.

ObjectivesLymphogranuloma venereum (LGV) is an STI caused by Chlamydia trachomatis serovars L1-L3. In Europe, the current epidemic is caused mainly by L2b genovariant, although increasing cases associated with other L2 variants have been reported. Here, we assessed the distribution of rectal LGV genovariants among men having sex with men (MSM) in Italy.MethodsFrom 2016 to 2020, all the anorectal swabs collected from MSM attending the STI Clinic of St. Orsola-Malpighi Hospital in Bologna and positive for C. trachomatis were stored. LGV infection was confirmed by a pmpH PCR, and, subsequently, a fragment of the ompA gene was amplified and sequenced. Sequences were aligned to reference strains representing different LGV variants.ResultsLGV cases accounted for one-third of all chlamydial rectal infections with a total prevalence of 4.1% (76/1852). Total number of LGV cases per year remained constant. LGV was mainly found in symptomatic patients (>65%), older than 30 years, with a high burden of other STIs (63.7% HIV-positive, 35.5% with concurrent rectal gonorrhoea, 19.7% with early syphilis). A decreasing trend in HIV-LGV co-infection was noticed over time. Three main LGV genovariants were detected (L2f, 46.1%; L2b, 23.0%; L2-L2b/D-Da, 16.9%), together with other known L2b variants (mainly L2bV2 and L2bV4). Two novel L2b ompA variants with non-synonymous single-nucleotide polymorphisms were found. Over time, the percentage of L2f cases dropped gradually, with a significant increase in L2-L2b/D-Da cases (p=0.04).ConclusionsIn our area, LGV is endemic among MSM with different circulating genovariants. Active surveillance and genotyping programmes are needed to reduce re-establishing of LGV infection.

Objectives: Metabolic syndrome (MetS) is usually associated in general population with systemic inflammation and higher cardiovascular risk, but data about the effect of statins in patients with HIV infection and MetS are lacking to date. Methods: Prospective cohort study of treated HIV-infected patients, aged from 40 to 60 years, with or without MetS, who started rosuvastatin (10 mg daily), and were followed-up for 12 months. The primary endpoint was change in serum levels of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α). The secondary endpoint was change in the carotid intima-media thickness (IMT). Results: One hundred and twenty-five patients were enrolled: 61 with MetS (MetS group) and 64 without MetS (control group). After 12 months, rosuvastatin produced a significant decrease in mean serum levels of hsCRP (-0.28 mg/dL; p = .037), IL-6 (-2.1 pg/mL; p = .018) and TNF-α (-6.3 pg/mL; p = .004) in patients with MetS. On the contrary, in controls rosuvastatin did not lead to a significant change in mean levels of all biomarkers. After 12 months, the mean IMT increase at the carotid bifurcation was significantly lower in the MetS group than in the control group at the carotid bifurcation (0.017 vs. 0.031 mm; p = .037) and in all other anatomical sites. Conclusion: Our findings suggest that rosuvastatin is effective in reducing serum inflammation markers and slowing atherosclerosis progression rate in HIV-infected patients on cART and with MetS, while its effects on serum biomarkers and IMT increase seem to be negligible in those without MetS.

Giardiasis, the disease caused by the flagellate Giardia duodenalis (syn. G.lamblia, G. intestinalis), is the most commonly reported among the five food- and waterborne parasitic diseases under mandatory surveillance in 24 EU countries. From November 2018 to April 2019, an outbreak of giardiasis occurred in a municipality of the Bologna province, in north-eastern Italy. Microscopy and immunochromatography identified cysts and antigens, respectively, of the parasite in stool samples of 228 individuals. Molecular typing of 136 stool samples revealed a vast predominance (95%) of G. duodenalis assemblage B. Investigations into potential sources indicated tap water as the most likely vehicle of infection, although cysts were not detected in water samples. Control measures mostly aimed at preventing secondary transmission by informing citizens about the outbreak, and by treatment of patients with anti-parasitic drugs. This is the first documented human outbreak of giardiasis in Italy; its investigation has highlighted the difficulties in the timely detection and management of this parasite, which is often overlooked as a cause of human gastroenteritis. The long and variable incubation time, absence of specific symptoms and a general lack of awareness about this pathogen contributed to delay in diagnosis.

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The oropharynx represents a crucial site for the emergence of multi-drug resistance in Neisseria gonorrhoeae. The mosaic penA alleles, associated with decreased susceptibility to cephalosporins, have emerged by DNA recombination with partial penA genes, particularly those from commensal pharyngeal Neisseria species. Here, we investigated the prevalence of the mosaic structure of the penA gene in the oropharynx of men who have sex with men testing negative for pharyngeal gonorrhoea. From January 2016 to June 2018, 351 gonorrhoea-negative men who have sex with men attending a sexually transmitted infection clinic in Italy were enrolled. Pharyngeal swabs underwent a real-time polymerase chain reaction (PCR) for the detection of the mosaic penA gene. In case of positivity, PCR products were sequenced and searched against several sequences of Neisseria strains. Overall, 31 patients (8.8%) were found positive for the presence of the mosaic penA gene. The positivity was significantly associated with previous cases of pharyngeal gonorrhoea (relative risk [RR]: 3.56, 95% confidence interval 1.44–8.80) and with recent exposure to beta-lactams (RR: 4.29, 95% confidence interval 2.20–8.38). All penA-positive samples showed a high relatedness (90–99%) with mosaic-positive Neisseria strains. Our data underline that commensal Neisseria species of the oropharynx may be a significant reservoir for genetic material conferring antimicrobial resistance in N. gonorrhoeae.

Hepatitis C virus (HCV) Core Antigen (HCVAg) and HCV-RNA were tested in 962 plasma/serum samples from 180 patients during Direct Antiviral Agents (DAAs) treatment and at follow-up. One hundred and eighty individuals were included: 71% carried advanced fibrosis and 43% were treatment-experienced. A Sustained Virological Response (SVR) was achieved in 166/180 (92%) individuals: 96/102 (94.1%) na ve and 70/78 (89.7%) treatment-experienced (p=0.20). The baseline median levels of HCV-RNA and HCVAg were not significantly different between individuals achieving SVR (5.92 x 105 IU/mL, IQR 5.4-6.4, and 3,417 fmol/L, 2,900-3,795) and those without SVR (6.06 x 105 IU/mL, 5.63-6.57, and 3,391 fmol/L, 2,828-4,077). The HCV-RNA vs. HCVAg assays results showed a fair correlation with an overall moderate qualitative agreement (kappa=0.52). Among treatment-failed individuals, at failure 100% of the assays results were positive for both techniques, with HCV-RNA median value 3.09 x 105 IU/mL (2.10-29.09) and HCVAg median value 1570.28 fmol/L (360.15-9317.67). Undetectable HCV-RNA at EOT showed sensitivity 54%, specificity 100%, negative predictive value (NPV) 93% and positive predictive value (PPV) 100%. Undetectable HCVAg at EOT showed sensitivity 74%, specificity 100%, NPV 97% and PPV 100%. The operative and economic advantages of the HCVAg support the alternative use of HCVAg to monitor DAAs treatment outcome.

Vaginal microbes and their metabolic products have crucial functions, affecting local immunity development and maternal-fetal health. The composition of the vaginal microbiome can vary in response to various factors, including body mass index (BMI), and diet. In this study we get new insights into the vaginal ecosystem of Caucasian women (n = 24) at the first trimester of pregnancy, assessing whether pre-pregnancy diet can affect the structure of the vaginal environment in terms of bacterial composition and vaginal metabolite concentration. We characterized 1) the vaginal bacterial composition (Nugent score), 2) the vaginal metabolic profiles (1H-NMR spectroscopy), and 3) the dietary nutrient intake by means of a validated food frequency questionnaire. Pre-pregnancy BMI was negatively related to vaginal health status, indicating that women who begin pregnancy overweight/obese have a greater occurrence of vaginal dysbiosis during pregnancy. A lactobacilli-dominated vaginal microbiota was negatively associated with higher pre-pregnancy intake of animal-sourced protein. Conversely, a higher pre-pregnancy consumption of total carbohydrates and sugars seemed to be a protective factor for vaginal health. The vaginal environment of BV-women was characterized by higher levels of biogenic amines and organic acids, whereas higher levels of phenylpropionate and diverse amino acids were fingerprints of a healthy vaginal status. A significant association between a higher pre-pregnancy BMI and several dysbiosis-related vaginal metabolites was also found. Our study shed light on the role of pre-pregnancy BMI and diet on the vaginal environment during pregnancy, underlining the importance of limiting protein intake from animal foods to maintain a healthy lactobacilli-dominated microbiota.

The SARS-CoV-2-associated COVID-19 pandemic has shaken the global healthcare system. Although the best-known symptoms are dry cough and pneumonia, viral RNA has been detected in the stool and about half of COVID-19 patients exhibit gastrointestinal upset. In this scenario, special attention is being paid to the possible role of the gut microbiota (GM). Fecal samples from 69 COVID-19 patients from three different hospitals of Bologna (Italy) were analyzed by 16S rRNA gene-based sequencing. The GM profile was compared with the publicly available one of healthy age- and gender-matched Italians, as well as with that of other critically ill non-COVID-19 patients. The GM of COVID-19 patients appeared severely dysbiotic, with reduced diversity, loss of health-associated microorganisms and enrichment of potential pathogens, particularly Enterococcus. This genus was far overrepresented in patients developing bloodstream infections (BSI) and admitted to the intensive care unit, while almost absent in other critically ill non-COVID-19 patients. Interestingly, the percentage of patients with BSI due to Enterococcus spp. was significantly higher during the COVID-19 pandemic than in the previous 3 years. Monitoring the GM of critically ill COVID-19 patients could help clinical management, by predicting the onset of medical complications such as difficult-to-treat secondary infections.

BACKGROUND: To date, data about prevalence and clinical impact of viral hepatitis in COVID-19 are scarce and conflicting.METHODS: We conducted an observational, retrospective study including all subjects tested positive for SARS-CoV-2 from March to May 2020. We evaluated prevalence of chronic viral hepatitis and we performed a matched cohort analysis of patients with and without chronic viral hepatitis comparing COVID-related outcomes.RESULTS: 980 subjects resulted positive to SARS-CoV-2 on oropharyngeal swabs. Among them 12 (1,2%) were HBsAg positive and 6 (0,6%) had detectable HCV RNA. No one was receiving antiviral therapy. None of these had a documented liver cirrhosis.We identified 80 SARS-CoV-2 positive individuals with negative viral markers for HBV and HCV for the matched analysis. No statistical differences in hospitalization, need for MV and mortality rates (p 0.79, p 0.28 and p 0.8, respectively) were found. Although not statistically significant (p 0.12), a higher rate of those positive for viral hepatitis were admitted to ICU (29% Vs 15%). Median time of virus clearance was 27.5 (IQR 20,38) days, with no difference between the two groups (p 0.39, 95% CI (-12;4.8)). We found older age and male sex as factors associated with worse outcomes.CONCLUSION: Our analysis documented similar rates of chronic viral hepatitis among subjects with SARS-CoV-2 infection and general population. Furthermore, in our population, pre-existing viral liver infection did not have any impact on the clinical and virological course of COVID-19.

Weight gain associated with integrase inhibitor-based treatment has become a critical issue in the clinical management of HIV infection. We analyzed changes in weight and body fat mass in 54 virologically suppressed patients who switched to lamivudine plus raltegravir or dolutegravir. Overall, after 12 months we reported a not significant increase in weight (median, +1.74 kg; p = .223) and total fat mass (median, +1.13 kg; p = .188), and these changes were comparable between groups. The median change in lumbar spine bone mineral density (BMD) [interquartile range (IQR)] was +0.02 g/cm2 (-0.02, +0.05; p = .786), and the median change in femur neck BMD (IQR) was +0.04 g/cm2 (-0.03, +0.06; p = .598), and changes were comparable between groups. In conclusion, the switch to dolutegravir/lamivudine or raltegravir/lamivudine dual therapy in virologically suppressed patients did not produce significant increases in weight and body fat mass after a 12-month follow-up, in association with not significant changes in BMD.

In most children, coronavirus disease 2019 (COVID-19) is a mild or moderate disease. Moreover, in a relevant number of cases, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains totally asymptomatic. All these findings seem to suggest that otherwise healthy children with suspected COVID-19 might be managed in the community in most cases, thus avoiding hospital admission and closely related medical, social and economic problems, including overwhelming hospitals. Unfortunately, home management of children with suspected COVID-19 rarely occurs, and many children with suspected or laboratory-confirmed SARS-CoV-2 infection are frequently hospitalized irrespective of the severity of disease. To evaluate the role of community health houses (CHHs) in the management of children with COVID-19, 1,009 children with suspected SARS-CoV-2 infection were studied in Emilia-Romagna Region, Italy. Among them, 194 (19.2%) resulted positive for SARS-CoV-2. The majority (583, 58%) were tested at home by CHHs, while 426 (42%) were brought to the hospital for testing. The patients who were managed in the hospital had a significantly lower median age than those who were managed at home (2 vs. 12 years, p < 0.001). Exposure to SARS-CoV-2 cases within the family was significantly more frequent among those who were managed at home (82 vs. 46%, p < 0.05). The clinical findings were similar between the children who were managed at home and those who were managed in the hospital. Only one of the children managed at home (0.7%) required hospitalization; in comparison, 26 (48%) of those whose swab samples were taken at the hospital were hospitalized. Our research shows for the first time the importance of CHHs in the management of COVID-19 in children; because of the high frequency of mild to moderate cases, management by CHHs can reduce the care load in hospitals, providing enormous advantages on the familial, medical, social, and economic levels. These findings could be useful for suggesting a territorial rather than hospital-based strategy in pediatrics in the case of a new wave of the epidemic.

Introduction: Neurological manifestations are emerging as relatively frequent complications of corona virus disease 2019 (COVID-19), including stroke and encephalopathy. Clinical characteristics of the latter are heterogeneous and not yet fully elucidated, while the pathogenesis appears related to neuroinflammation in a subset of patients. Case: A middle-aged man presented with acute language disturbance at the emergency department. Examination revealed expressive aphasia, mild ideomotor slowing, and severe hypocapnic hypoxemia. Multimodal CT assessment and electroencephalogram (EEG) did not reveal any abnormalities. COVID-19 was diagnosed based on chest CT findings and positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription PCR (RT-PCR) on nasopharyngeal swab. The following day, neurological symptoms progressed to agitated delirium and respiratory status worsened, requiring admission to the ICU and mechanical ventilation. Brain MRI and cerebrospinal fluid (CSF) studies were unremarkable. RT-PCR for SARS-CoV-2 on CSF was negative. He received supportive treatment and intravenous low-dose steroids. His neurological and respiratory status resolved completely within 2 weeks. Conclusions: We report a patient with reversible COVID-19-related encephalopathy presenting as acute aphasia, mimicking stroke or status epilepticus, eventually evolving into delirium. Although large-vessel stroke is frequently encountered in COVID-19, our case suggests that focal neurological deficits may occur as the earliest feature of encephalopathy. Neurological status reversibility and the absence of abnormalities on brain MRI are consistent with a functional rather than a structural neuronal network impairment.

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Toscana virus (TOSV) is a Phlebovirus transmitted by phlebotomine sand flies and is an important etiological agent of summer meningitis in the Mediterranean basin. Since TOSV infection is often asymptomatic, we evaluated the seroprevalence in blood donors (BDs) in the Bologna and Ferrara provinces (Northeastern Italy)&mdash, the areas with the highest and lowest numbers of TOSV neuroinvasive cases in the region, respectively. A total of 1208 serum samples from BDs were collected in April&ndash, June 2014 and evaluated for the presence of specific TOSV-IgG by ELISA. The IgG-reactive samples were confirmed by indirect immunofluorescence assay (IIF) and by microneutralization test (MN). Serum samples were defined as positive for anti-TOSV IgG when reactive by ELISA and by at least one second-level test, TOSV seroprevalence was 6.8% in the Bologna province, while no circulation of TOSV was detected in the Ferrara province. Sand fly abundance in 2014 was also estimated by a geographic information system using a generalized linear model applied to a series of explanatory variables. TOSV seroprevalence rate was strongly associated with the sand fly abundance index in each municipality, pointing out the strong association between sand fly abundance and human exposure to TOSV.

The population structure of human isolates of Listeria monocytogenes in Emilia-Romagna, Italy, from 2012 to 2018 was investigated with the aim of evaluating the presence of genomic clusters indicative of possible outbreaks, the proportion of cluster-associated vs. sporadic isolates and different methods and metrics of genomic analysis for use in routine surveillance. In the 2012–2018 period the notification rate of confirmed invasive cases in Emilia-Romagna was 0.91 per 100,000 population per year, more than twice the average rate of EU countries. Out of the total 283 cases, 268 (about 95%) isolates were typed through whole genome sequencing (WGS) for cluster detection with methods based on core-genome multi-locus sequence typing and single nucleotide polymorphisms. Between 66 and 72% of listeriosis cases belonged to genomic clusters which included up to 27 cases and lasted up to 5 years. This proportion of cluster-associated cases is higher than previously estimated in other European studies. Rarefaction analysis, performed by reducing both the number of consecutive years of surveillance considered and the proportion of isolates included in the analysis, suggested that the observed high proportion of cluster-associated cases can be ascribed to the long surveillance duration (7 years) and the high notification and typing rates of this study. Our findings show that a long temporal perspective and high surveillance intensity, intended as both exhaustiveness of the system to report cases and high WGS-typing rate, are critical for sensitive detection of possible outbreaks within a WGS-based surveillance of listeriosis. Furthermore, the power and complexity of WGS interpretation emerged from the integration of genomic and epidemiological information in the investigation of few past outbreaks within the study, indicating that the use of multiple approaches, including the analysis of the accessory genome, is needed to accurately elucidate the population dynamics of Listeria monocytogenes.

Background Antiretroviral dual regimens including lamivudine and one boosted PI or dolutegravir are warranted in order to optimize combination ART (cART), prevent long-term toxicity and reduce the cost of treatments. Objectives We hypothesized that a maintenance dual regimen of lamivudine plus raltegravir would be effective and as well tolerated as the dual maintenance combination of lamivudine plus dolutegravir. Methods We performed an observational, retrospective study of HIV-infected patients on suppressive ART who switched to a dual regimen containing lamivudine 300 mg once daily plus raltegravir 1200 mg once daily or dolutegravir 50 mg once daily. Results In total, 109 patients (79 men; mean age 46.4 years; mean CD4+ T lymphocyte count 605 cells/mm3) were enrolled. Overall, 50 subjects switched to lamivudine plus raltegravir (Group A) and 59 to lamivudine plus dolutegravir (Group B). After 12 months, 45 patients (90%) in Group A and 52 (88.1%) in Group B had HIV RNA Conclusions In our study, dual therapies containing lamivudine plus raltegravir or dolutegravir in virologically suppressed patients showed high and comparable efficacy, as well as good tolerability.

Objectives Active screening is a crucial element for the prevention of carbapenemase-producing Enterobacteriaceae (CPE) transmission in healthcare settings. Here we propose a culture-based protocol for rectal swab CPE screening that combines CPE detection with identification of the carbapenemase type. Methods The workflow integrates an automatic digital analysis of selective chromogenic media (WASPLab®; Copan), with subsequent rapid tests for the confirmation of carbapenemase production [i.e. detection of Klebsiella pneumoniae carbapenemase (KPC)-specific peak by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) or a multiplex immunochromatographic assay identifying the five commonest carbapenemase types]. To evaluate the performance of this protocol in depth, data for 21 162 rectal swabs submitted for CPE screening to the Microbiology Unit of S. Orsola-Malpighi Hospital (Bologna, Italy) were analysed. Results Considering its ability to correctly segregate plates with/without Enterobacteriaceae, WASPLab Image Analysis Software showed globally a sensitivity and specificity of 100% and 79.4%, respectively. Of the plates with bacterial growth (n = 901), 693 (76.9%) were found to be positive for CPE by MALDI-TOF/MS (KPC-specific peak for K. pneumoniae) or by immunochromatographic assay. Only 2.8% (16/570) of KPC-positive K. pneumoniae strains were missed by the specific MALDI-TOF/MS algorithm, being detected by the immunochromatographic assay. The mean turnaround time needed from sample arrival to the final report ranged between 18 and 24 h, representing a significant time saving compared with manual reading. Conclusion This workflow proved to be fast and reliable, being particularly suitable for areas endemic for KPC-producing K. pneumoniae and for high-throughput laboratories.

A better characterization of the HIV reservoir is pivotal for the development of effective eradication strategies. Accurate quantification of the latent reservoir remains challenging. Starting from a regular blood draw, the Tat/Rev induced limiting dilution assay (TILDA) combines serial dilution of CD4+ T cells with a PCR-based detection of HIV-1 spliced mRNA produced upon cell stimulation. Here we adapted the original protocol for HIV-1 subtype B to detect tat/rev mRNAs transcribed from reactivated latently infected cells in long term suppressed patients infected with HIV-1 subtype C. Given the heterogeneity of global HIV epidemiology, it is pivotal to develop assays with optimal performances also in patients infected with non-B subtypes. We observed that, in these patients infected with subtype C virus, the HIV reservoir quantified by TILDA correlates with both the time of virological suppression and CD4/CD8 ratio.

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent the most common agents of sexually transmitted rectal infections among men having sex with other men (MSM). In this study, we assessed the bacterial composition of the rectal microbiota associated with CT and/or NG infections in a cohort of men reporting unsafe rectal intercourse. A total of 125 rectal swabs were collected and four groups were compared: non-infected subjects (n = 53), patients with CT (n = 37), or NG rectal infection (n = 17) and patients with contemporary positivity for CT/NG (n = 18). CT and NG infections were detected by a real-time commercial test and the rectal microbiota composition was analyzed from rectal swabs through sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene. The rectal microbiota of all subgroups was dominated by Prevotellaceae, Enterobacteriaceae, and Ruminococcaceae families. Irrespective of the analyzed subgroup, we found that the rectal environment of all the enrolled MSM was rich in Prevotella and Escherichia genera. Moreover, a shift in the bacterial composition between patients with sexually transmitted rectal infections and controls was noticed: infected patients were characterized by a depletion of Escherichia species, associated with an increase of anaerobic genera, including Peptoniphilus, Peptostreptococcus, and Parvimonas. Overall, the presence of rectal symptoms did not significantly modify the rectal microbiota profiles among the four groups of analyzed patients. We confirmed that HIV-positive patients are characterized by a lower bacterial richness than HIV-negative subjects. However, we found that the presence of HIV has a different impact on bacterial rectal communities compared to CT and NG infections, modifying the relative abundance of several genera, including Gardnerella, Lactobacillus, Corynebacterium, and Sutterella. Information about the rectal microbiota composition in CT and NG infections could shed light on the pathogenesis of these conditions and could contribute to the onset of new strategies for their control.

New strategies for HIV treatment able to reduce drug-exposure, medium-long term toxicities and costs are a recognized clinical need. The availability of newer drugs with improved potency and tolerability, as well as high genetic barrier to resistance, makes antiretroviral-sparing strategies with two-drug regimens (2DRs) particularly attractive. Substantial evidence has been generated over the last few years supporting 2DR in virologically suppressed HIV infected patients for whom a therapy switch is planned. More recently, very promising data on 2DR in naïve patients have also been reported. The main purpose of this consensus is to provide an overview of guideline indications and recommendations, and the most recent data from clinical studies of 2DR in both naïve and virologically suppressed patients. As an expert consensus, suggestions and indications on the use and management of 2DR are also provided.

Combination therapies are frequently used in the treatment of multidrug-resistant Klebsiella pneumoniae infection without consensus regarding which combination is the most effective. We compared bactericidal titres from sera collected from critically ill patients receiving meropenem plus tigecycline (n= 5), meropenem plus colistin (n= 5), or meropenem, colistin and tigecycline (n= 5) against K. pneumoniae isolates that included ESBL-producing (n= 7) and KPC-producing strains (n= 14) with varying sensitivity patterns to colistin and tigecycline. Meropenem concentrations (Cmin) were measured in all samples by LC-MS/MS, and indexed to respective pathogen MICs to explore differences in patterns of bactericidal activity for two versus three drug combination regimens. All combination regimens achieved higher SBTs against ESBL (median reciprocal titre 128, IQR 32–256) versus KPC (4, IQR 2–32) strains. Sera from patients treated with meropenem-colistin yielded higher median SBTs (256, IQR 64–512) than either meropenem-tigecycline (32, IQR 8–256; P' 0.001). The addition of tigecycline was associated with a lower probability of achieving a reciprocal SBT above 8 when meropenem concentrations were below the MIC (P= 0.04). Although the clinical significance is unknown, sera from patients receiving tigecycline-based combination regimens produce lower serum bactericidal titres against ESBL or KPC-producing K. pneumoniae. SBTs may represent a useful complimentary endpoint for comparing pharmacodynamics of combinations regimens for MDR Enterobacteriaceae.

In this study we assessed the prevalence and predictors of pharyngeal infections due to Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in a cohort of patients attending an STI Clinic. From January 2016 to September 2018, 893 patients attending the STI Clinic of St Orsola-Malpighi Hospital (Bologna, Italy) and reporting unprotected oral sex were enrolled. A pharyngeal swab for the molecular detection of CT and NG was collected from each patient. Positive CT samples were further genotyped by an omp1 gene PCR. A total of 134 cases of gonorrhea (15%) and 34 chlamydial infections (3.8%) were detected in the pharyngeal site, with no significant differences between males (all men who have sex with men [MSM]) and females. More than 90% of the infections were completely asymptomatic, suggesting that symptoms were not predictors of a pharyngeal infection ( P = 0.7). On the contrary, a history of sexual contacts with a partner positive for CT and/or NG ( P

This study compares the performance of seven assays, including two ELISA (Leishmania ELISA IgG + IgM, Vircell Microbiologists; Leishmania infantum IgG ELISA, NovaTec), three rK39-based immunochromatographic tests (rK39-ICTs) (Leishmania Dipstick Rapydtest, Apacor; On Site Leishmania IgG/IgM Combo Rapid Test, CTK Biotech; LEISHMANIA Strip quick Test, Cypress Diagnostic), one indirect immunofluorescent antibody test (IFAT) (Leishmania-Spot IF, BioMérieux), and one western blot (WB) (Leishmania WESTERN BLOT IgG, LDBio Diagnostics) for serodiagnosis of visceral leishmaniasis (VL). Serum samples from 27 VL patients living in northeastern Italy were analyzed, as well as the serum samples from 50 individuals in whom VL diagnosis was excluded. The WB and the IFAT had 96% sensitivity, followed by the ELISA (63% and 74%, respectively). The rK39-ICT exhibited the worst performance among the serological tests, with sensitivities ranging from 52% to 70%. By combining selected ELISA/ICT, the sensitivity of VL detection reached 89%. IFAT and WB outperformed ELISA and rK39-ICT by possessing optimal sensitivity, but their high cost and complexity of execution would not allow their employment as screening tests. In conclusion, the combination of easy-to-perform tests, such as ICT and ELISA, could improve sensitivity in the serodiagnosis of Mediterranean VL.

Chlamydia persistence is a viable but non-replicative stage, induced by several sub-lethal stressor agents, including beta-lactam antibiotics. So far, no data about the connection between doxycycline and chlamydial persistence has been described in literature. We investigated the ability of doxycycline to induce C. trachomatis (CT) persistence in an in vitro model of epithelial cell infection (HeLa cells), comparing the results with the well-established model of penicillin-induced persistence. The effect of doxycycline was explored on 10 different CT strains by analysing (i) the presence of aberrant inclusions, (ii) chlamydial recovery, (iii) the expression of different chlamydial genes (omcB, euo, Ct110, Ct604, Ct755, HtrA) and (iv) the effects on epithelial cell viability. For each strain, the presence of foreign genomic islands responsible of tetracycline resistance was excluded. We found that low doses of doxycycline can induce a condition of CT persistence. For concentrations of doxycycline equal to 0.03–0.015 mg/L, CT inclusions are smaller and aberrant and CT cycle is characterized by the presence of viable but non-dividing RBs with the complete abolishment of chlamydial cytotoxic effect. Infectious EBs can be recovered after removal of the drug. During doxycycline-induced persistence, the expression of the late gene omcB is decreased, indicating the blocking of RB-to-EB conversion. Conversely, as for penicillin G, a significant up-regulation of the stress response HtrA gene is found in doxycycline-treated cells. This study provides a novel in vitro cell model to examine the characteristics of doxycycline-induced persistent CT infection.

Objectives The aim of this study was to evaluate the incidence of ceftazidime/avibactam resistance among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) strains isolated from patients with bloodstream infection. Methods We collected 120 carbapenemase producing Enterobacteriaceae (CPE) strains from unique patients hospitalized in two Italian hospitals between January 2018 to February 2019. Strains were phenotypically characterized for the type of carbapenemase production and susceptibility to ceftazidime/avibactam. Ceftazidime/avibactam-resistant strains were characterized by whole-genome sequencing. Results During the study period, we characterized 105 (87.5%) KPC producers among a total of 120 CPE strains. Ceftazidime/avibactam resistance was found in three KPC-Kp strains isolated from patients with no history of previous ceftazidime/avibactam-based treatment. Of note, two out of three ceftazidime–avibactam-resistant KPC-Kp were also resistant to meropenem/vaborbactam. Genomic characterization showed that a ceftazidime/avibactam-resistant KPC-Kp harboured a mixed population with D179Y mutated KPC-2, while the other two ceftazidime–avibactam-resistant KPC-Kp possessed non-functional ompK35-ompK37 and mutated ompK36 porins associated with higher copy number of blaKPC gene. Conclusions Our results showed that incidence of ceftazidime/avibactam resistance emerged in KCP-Kp strains independently from previous antimicrobial exposure. Resistance to ceftazidime/avibactam was associated with mutations within the blaKPC gene or porin deficiency associated with higher blaKPC copy number and is also related to the meropenem/vaborbactam resistance.

Mammalian cells have evolved several mechanisms to prevent or block lentiviral infection and spread. Among the innate immune mechanisms, the signaling cascade triggered by type I interferon (IFN) plays a pivotal role in limiting the burden of HIV-1. In the presence of IFN, human cells upregulate the expression of a number of genes, referred to as IFN-stimulated genes (ISGs), many of them acting as antiviral restriction factors (RFs). RFs are dominant proteins that target different essential steps of the viral cycle, thereby providing an early line of defense against the virus. The identification and characterization of RFs have provided unique insights into the molecular biology of HIV-1, further revealing the complex host-pathogen interplay that characterizes the infection. The presence of RFs drove viral evolution, forcing the virus to develop specific proteins to counteract their activity. The knowledge of the mechanisms that prevent viral infection and their viral counterparts may offer new insights to improve current antiviral strategies. This review provides an overview of the RFs targeting HIV-1 replication and the mechanisms that regulate their expression as well as their impact on viral replication and the clinical course of the disease.

Background The new Xpert MTB/RIF Ultra assay (Ultra, Cepheid, Sunnyvale, USA) is a cartridge-based automated diagnostic test that can simultaneously identify Mycobacterium tuberculosis complex (MTB) and resistance to Rifampicin (RIF). With respect to the previous version Xpert MTB/RIF assay (Xpert), IS6110/IS1081 repetitive elements probes have been added allowing the detection of lower MTB load, defined by the new semi-quantitative category “trace” with indeterminate RIF resistance. The aim of this study was to evaluate performance of the new version Ultra on Xpert-negative, but TB culture-positive clinical samples. Methods The de-identified frozen samples (-20 °C) collected over a 4-year period (February 2014-October 2017), which had previously resulted smear-negative, Xpert-negative but MTB culture-positive, were analyzed with Ultra. The de-frosted samples were loaded into the cartridge using the same process as the previous version, according to manufacturer’s instruction. Results During the study period 382 MTB culture-positive samples were archived: 314 resulted Xpert-positive and 68 Xpert-negative. Thirty-one of the 68 Xpert-negative samples resulted positive with Ultra, with an overall improvement in MTB detection of 45.6%. Out of 36 Xpert-negative respiratory samples, 18 resulted Ultra-positive with the following semi-quantitative loads: “low”(n = 1), “very low”(n = 11), “trace”(n = 6), with an improvement in MTB detection of 50%. The best performance was achieved on bronchoalveolar lavage specimens (53.8%). Out of 32 Xpert-negative non-respiratory samples, 13 resulted Ultra-positive with the following semi-quantitative loads: “very low”(n = 7), “trace”(n = 6), with an improvement in MTB detection of 40.6%. The best performance was achieved on biopsies (55.6%) and lymph nodes (50%). The new category “trace” detected 12 out of the 31 Ultra-positive MTB samples; in the remaining 19 samples RIF susceptibility was determined with 100% concordance with the phenotypic susceptibility test. The mean time to positivity of samples found negative by Ultra was significantly longer in comparison to positive samples in liquid culture. Conclusions Our results are consistent with the few studies published so far and confirm the better performance of Ultra compared to the previous version in both respiratory and non-respiratory smear-negative samples, with an overall improvement of 45.6%.

Background: Nucleoside reverse transcriptase inhibitor (NRTI)-sparing antiretroviral therapies may be useful in HIV-infected patients with resistance or intolerance to this class. Methods: We performed an observational study of patients on suppressive antiretroviral therapy containing two NRTIs plus one ritonavir-boosted protease inhibitor who switched to a dual regimen containing raltegravir (400 mg twice daily) and darunavir/ritonavir (800/100 mg once daily) and were followed-up for 48 weeks. Results: As a whole, 82 patients were enrolled. Mean duration of current regimen was 4.6 years and mean duration of plasma HIV RNA < 50 copies/mL before the switch was 46.2 months. Reason for simplification was toxicity in 76 patients and resistance to NRTIs in 13. After switching, the percentage of patients with HIV RNA < 50 copies/mL at week 48 was 92.7% in the intent-to-treat-exposed analysis and 97.6% in the per-protocol analysis. The switch led to a significant reduction in the mean triglyceride value (-85.2 mg/dL), in the prevalence of tubular proteinuria (-56%) and in the mean level of interleukin-6 (-0.94 pg/mL), with a significant increase in the mean phosphoremia (+0.58 mg/dL). Mean trough concentrations of both raltegravir and darunavir were within the therapeutic range. Two patients (2.4%) had virological failure due to suboptimal adherence and 4 subjects (4.9%) discontinued treatment due to adverse events, but no patients experienced Grade 3 or 4 adverse events. Conclusion: In our study, simplification to a dual therapy containing raltegravir plus darunavir/ritonavir after 48 weeks maintained viral suppression in more than 90% of patients and showed a good tolerability with a favourable effect on proteinuria, ipophosphoremia, and lipid metabolism.

Objectives Recent clinical studies and one meta-analysis have shown a modest but significant increase in the incidence of diabetes mellitus associated with statin exposure, so this correlation was investigated in a cohort of HIV-positive subjects. Methods A retrospective cohort study including adult HIV-1-infected patients followed at our Clinic of Infectious Diseases between 2007 and 2014 was performed. Results We assessed 3170 HIV-positive patients with a median follow-up of 5.2 years. The incidence of diabetes mellitus was 1.2 per 100 person-years and it was not significantly associated with the prescription of statins [hazard ratio (HR) 1.09 per year of statin exposure; 95% confidence interval (CI) 0.7–1.49; P = 0.067], while it was associated with older age, chronic hepatitis C, antiretroviral-naive vs. antiretroviral experienced condition, high body mass index, and high serum concentration of triglycerides. Conclusions In our study, a higher risk of diabetes mellitus was not associated with statin treatment, but with some traditional risk factors.

Background: The ritonavir-boosted protease inhibitor (PI/r)-based dual regimens are warranted in order to optimize the combination antiretroviral therapy (cART), prevent the long-term toxicity and reduce the cost of treatments. Methods: We performed an observational, retrospective study of HIV-infected patients on suppressive antiretroviral therapy who switched to a dual regimen containing lamivudine (3TC) plus darunavir/ritonavir (DRV/r) 800/100 mg qd or atazanavir/ritonavir (ATV/r) 300/100 mg qd. Results: As a whole, 122 well-treated patients (mean age, 45.2 years; mean CD4 T + lymphocyte count, 589 cells/mm3; mean duration of current cART, 3.1 years) were enrolled. Current antiretroviral regimen included tenofovir/emtricitabine in 91 subjects, abacavir/lamivudine in 25, lopinavir/r in 41, DRV/r in 38 and ATV/r in 33. Baseline mean estimated glomerular filtration rate (eGFR) was 94.2 mL/min/1.73 m2, and proteinuria was detected in 46 subjects (38%). Overall 70 subjects switched to 3TC + DRV/r (group A) and 52 to 3TC + ATV/r (group B). After 12 months, 65 patients (92.8%) in group A and 46 (88.4%) in group B showed HIV RNA 

Pharyngeal gonorrhoea is a common sexually transmitted infection among 'men having sex with other men' (MSM). Neisseria gonorrhoeae (NG) pharyngeal infections are usually characterized by the absence of symptoms, acting as an important reservoir for their further spread. To the best of our knowledge, no information about the composition of the pharyngeal microbiome during an ongoing NG infection is currently available. Therefore, in this study, we characterized the pharyngeal bacterial community profiles associated with NG infection in a well-selected cohort of HIV-negative MSM reporting unsafe oral intercourse. A total of 70 pharyngeal swabs were considered, comparing non-infected subjects (n = 45) versus patients with pharyngeal gonorrhoea (n = 25) whose microbiota composition was analyzed from pharyngeal swabs through sequencing of hypervariable V3-V4 regions of the 16S rRNA gene. The pharyngeal microbiome of all subjects was dominated by Prevotellaceae, Veillonellaceae and Streptococcaceae families. Patients with pharyngeal gonorrhoea harboured a pharyngeal microbiome quite similar to negative subjects. Nevertheless, when looking to less-represented bacterial species (relative abundance approximately 1% or less), an imbalance between aerobe and anaerobe microorganisms was observed in NG-infected patients. In particular, the pharyngeal microbiome of NG-positive individuals was richer in several anaerobes (e.g. Treponema, Parvimonas, Peptococcus, Catonella, Filifactor) and poorer in various aerobe genera (i.e. Pseudomonas, Escherichia), compared to non-infected controls. No significant differences were noticed in the distribution of commensal Neisseria species of the oropharynx between NG-positive and negative subjects. Metabolic variations induced by changes in the microbiome abundance were assessed by a functional prediction of the bacterial metabolic pathways: a more abundant involvement of D-glutamine and D-glutamate metabolism, carbohydrate metabolism, as well as a greater activation of the energy metabolism was observed in patients with pharyngeal gonorrhoea compared to non-infected individuals. Information about the bacterial composition of the pharyngeal microbiome in case of gonorrhoea could shed light on the pathogenesis of the infection and open new perspectives for the prevention and control of this condition.

Although an independent evolution of viral quasispecies in different body sites might determine a differential compartmentalization of viral variants, the aim of this paper was to establish whether sequences from peripheral blood mononuclear cells (PBMCs) and plasma provide different or complementary information on HIV tropism in patients with acute or chronic infection. Tropism was predicted using genotypic testing combined with geno2pheno (coreceptor) analysis at a 10% false positive rate in paired RNA and DNA samples from 75 antiretroviral-naive patients (divided on the basis of avidity index into patients with a recent or long-lasting infection). A high prevalence of R5 HIV strains (97%) was observed in both compartments (plasma and PBMCs) in patients infected recently. By contrast, patients with a long-lasting infection showed a quite different situation in the two compartments, revealing more (46%) X4/DM in PBMCs than patients infected recently (3%) (P = 0.008). As- a knowledge of viral strains in different biological compartments might be crucial to establish a therapeutic protocol, it could be extremely useful to detect not only viral strains in plasma, but also viruses hidden or archived in different cell compartments to better understand disease evolution and treatment efficacy in patients infected with HIV. J. Med. Virol. 87:315–322, 2015. © 2014 Wiley Periodicals, Inc.

Kidney disease represents an important health concern among HIV-infected individuals, with an estimated prevalence ranging between 2.4 and 17%. The widespread use of antiretroviral drugs has changed the epidemiology of kidney disease in the HIV positive population, drastically reducing the percentage of patients affected by HIV-associated nephropathy (HIVAN), a complication characterized by apoptosis and de-differentiation of renal epithelial cells and podocytes. However, impaired kidney function remains an important issue among HIV-infected patients because of their long-term exposure to antiretroviral drugs and the growing burden of traditional risk factors associated with chronic renal disease. Furthermore, since HIV infects renal epithelial cells, kidney is a potential reservoir site that needs to be considered in future eradication studies. This review summarizes the main risk factors associated with chronic kidney disease in HIV-infected patients and discusses the contribution of viral infection and antiretroviral therapy to the pathogenesis of renal damage, emphasizing the need to monitor kidney status during the follow-up of HIV-infected patients.

Background Several studies have shown a significant association between vitamin D deficiency and an increased risk of statin-related symptomatic myalgia in the general population, but there are no data among HIV-infected persons. Methods A retrospective, cohort study was conducted to assess the incidence of symptomatic myalgia and elevation in serum creatine kinase level among HIV-positive adults on combination antiretroviral therapy and treated with atorvastatin or rosuvastatin for at least 12 months between 2011 and 2015 in our outpatient unit. Results A total of 545 patients (mean age 53.4 years) were enrolled into the study. Atorvastatin was prescribed in 55.8% of patients and rosuvastatin in 44.2%. After a mean duration of statin therapy of 29 months, an isolated symptomatic myalgia was diagnosed in 42 patients (7.7%) and a myalgia associated with elevated creatine kinase level in 25 (4.6%). The mean concentration of 25-hydroxyvitamin D was significantly lower in patients with myalgia (19.4 ng/ml) and with creatine kinase elevation and myalgia (22.8 ng/ml) than in those without muscle toxicity (32.1 ng/ml; P = 0.017 and 0.024, respectively). In stratified multivariable-adjusted logistic regression models, there was a statistically significant association between vitamin D deficiency and occurrence of symptomatic myalgia (P = 0.009) or creatine kinase elevation and myalgia (P = 0.046). Other factors significantly associated with development of myalgia were duration of statin therapy more than 24 months, history of myalgia, and age older than 60 years. Discussion In our observational study, vitamin D deficiency was significantly associated with a statin-induced myalgia among HIV-infected patients on combination antiretroviral therapy, in conformity with data of the general population.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Its clinical course is typically indolent; however, based on a series of pathobiological, clinical, genetic, and phenotypic parameters, patient survival varies from less than 5 to more than 20 years. In this paper, we show for the first time that the expression of the interferon-inducible DNA sensor IFI16, a member of the PYHIN protein family involved in proliferation inhibition and apoptosis regulation, is associated with the clinical outcome in CLL. We studied 99 CLLs cases by immunohistochemistry and 10 CLLs cases by gene expression profiling. We found quite variable degrees of IFI16 expression among CLLs cases. Noteworthy, we observed that a reduced IFI16 expression was associated with a very poor survival, but only in cases with ZAP70/CD38 expression. Furthermore, we found that IFI16 expression was associated with a specific gene expression signature. As IFI16 can be easily detected by immunohistochemistry or flow cytometry, it may become a part of phenotypic screening in CLL patients if its prognostic role is confirmed in independent series.

A 30-year-old man was admitted to hospital after complaining of a retrosternal burning pain, radiating to the jugular region, and to both upper limbs. An electrocardiography examination showed a ST segment elevation involving the lower-lateral leads. A trans-thoracic ultrasonography showed findings compatible with an acute myopericarditis. All performed serological testings excluded other recent infections with cardiac tropism. Among screening tests, a peripheral lymphocyte subset analysis was performed and an inversion of the CD4/CD8 ratio was found. Therefore, HIV testing was performed and proved positive for HIV-1 antibodies. The discovery of a primary HIV infection with involvement of a vital organ led us to start HAART. On day 20, our patient underwent a right heart catheterization and endomyocardial biopsy. During the following days, the clinical conditions of our patient improved, and a further heart ultrasonography documented a mild pericardial thickening as a result of the recent myopericarditis. Also the evolving changes of ECG were compatible with a benign evolution of myopericarditis. The histopathologic studies revealed a mild fibrosis of the myocardial right ventricular tissue, and inflammatory findings compatible with a recent myocarditis. At the real-time PCR analysis on bioptic sample, only HHV6 DNA and HIV-DNA were reactive. An immunofluorescence staining was performed to highlight the HIV p24 protein and a positive signal was detected in myocardial tissue. Considering the low avidity level of the anti-HIV IgG antibodies and the positivity of HIV-DNA in the endomyocardial tissue, we believe that the clinical manifestation presented can be referred to the recent primary HIV-infection.

In addition to immune system, HIV infection can determine the onset of functional dysfunctions of several organs and tissues such as central nervous system, cardiovascular system, kidney, and bone. It is noteworthy that HIV-infected individuals show an increased risk of bone mass loss with subsequent osteopenia and osteoporosis development. Interestingly, antiretroviral therapy is not able to tackle the bone derangement but, on the contrary, it can induce a progressive bone mass loss, especially when specific antiretroviral drugs are used. In this report, we summarized the HIV and antiretroviral-related mechanisms involved in the osteopenia and osteoporosis induction.

Background and aims HIV‐1 RNA viral load (VL) in plasma samples of HIV‐1–positive patients is used to assess the level of viral replication, the risk of disease progression, and the response and efficacy to antiretroviral treatment. Knowing the performance of different tests for HIV‐1 RNA detection is, therefore, important for clinical care. This study compared the performance of the recently introduced Aptima HIV‐1 Quant Dx assay (Hologic, Inc) and the standard COBAS AmpliPrep/COBAS TaqMan HIV‐1 v2.0 Test (CAP/CTM2) (Roche Molecular System, Inc) for HIV‐1 RNA quantitation. Methods Assay performance was assessed using 335 clinical samples, a standard HIV‐1 low VL panel, and 2 diluted samples from well‐characterized patients infected with different HIV‐1 subtypes tested in 5 replicates over 3 days. All samples were tested on both assays to evaluate inter‐assay agreement, both qualitatively and quantitively. Altogether, we evaluated assay sensitivity, linearity, accuracy, precision, repeatability, and reproducibility. Results Assay agreement for qualitative results in 335 clinical samples was fair (80.6%). Correlation of quantitative assay results (n = 164) was excellent (R 2 = 0.97), with 96.3% of the results within the 95% limit of assay agreement (−0.42 to +0.86 log), and 98.8% within 1 log of each other. Aptima‐HIV‐1 yielded results, on average, 0.22 log higher than CAP/CTM2. Both assays accurately quantitated the HIV‐1 standard at low VL (R 2 ≥ 0.94), with all samples within 0.5 log of the target. Conclusion Aptima‐HIV‐1 assay demonstrated sensitivity, accuracy, reproducibility, and precision for the detection and quantitation of HIV‐1 RNA across a wide dynamic range of VLs. Its performance, together with full automation and high throughput, suggests that Aptima‐HIV‐1 could be a suitable assay for reliable monitoring of HIV‐1 VL in patients undergoing treatment.

Several HIV-1 infected patients show bone loss and osteopenia/osteoporosis during the course of disease. The mechanisms underlying this degenerative process are largely unsettled and it has not been determined yet whether bone dysfunction is linked to HIV-1-mediated direct and/or indirect effects on osteoblasts/osteoclasts cross-talk regulation. This study investigated the effects of HIV-1(IIIb) and HIV-1(ADA) strains on osteoblasts using the osteoblast-derived cell line (HOBIT) and primary human osteoblasts as cellular models. The challenge of these cell cultures by both HIV-1 strains triggered a significant apoptosis activation unrelated to viral infection, since proviral HIV-1 DNA and supernatant HIV-1 RNA were not detected by real time PCR or b-DNA assays respectively. Under the experimental conditions, even heat-inactivated HIV-1 or cross-linked recombinant gp120 treatment of HOBIT and osteoblasts induced programmed cell death, suggesting that apoptosis is regulated by the interaction between HIV-1 gp120 and cell membrane. The analysis of cell culture supernatants showed a significant up-regulation of TNFalpha, a pleiotropic protein considered an apoptosis inducer in the osteoblast model. In fact, pretreatment of HOBIT and osteoblast cell cultures with anti-TNFalpha polyclonal antibody tackled effectively HIV-1 related induction of cell apoptosis. As a whole, these results indicate that HIV-1 may impair bone mass structure homeostasis by TNFalpha regulated osteoblast apoptosis.

none 6 no The introduction of potent combination antiretroviral therapy (cART) has had a remarkable impact on the natural history of HIV infection, leading to a dramatic decline in the mortality rate and a considerable increase in the life expectancy of HIV-positive people. However, cART use is frequently associated with several metabolic complications, mostly represented by lipid metabolism alterations, which are reported very frequently among persons treated with antiretroviral agents. In particular, hyperlipidaemia occurs in up to 70%-80% of HIV-positive subjects receiving cART and is mainly associated with specific antiretroviral drugs belonging to three classes of antiretroviral agents: NRTIs, NNRTIs and PIs. The potential long-term consequences of cART-associated dyslipidaemia are not completely understood, but an increased risk of premature coronary heart disease has been reported in HIV-infected patients on cART, so prompt correction of lipid metabolism abnormalities is mandatory in this population. Dietary changes, regular aerobic exercise and switching to a different antiretroviral regimen associated with a more favourable metabolic profile are the first steps in clinical management, but lipid-lowering therapy with fibrates or statins is often required. In this case, the choice of hypolipidaemic drugs should take into account the potential pharmacokinetic interactions with many antiretroviral agents. mixed Calza, Leonardo; Colangeli, Vincenzo; Manfredi, Roberto; Bon, Isabella; Re, Maria Carla; Viale, Pierluigi Calza, Leonardo; Colangeli, Vincenzo; Manfredi, Roberto; Bon, Isabella; Re, Maria Carla; Viale, Pierluigi

In this study, we showed the existence of a positive correlation between the amount of human immunodeficiency virus-type 1 (HIV-1) RNA in HIV-1 seropositive subjects and the plasma levels of TRAIL. Since it has been previously demonstrated that HIV-1 Tat protein up-regulates the expression of TRAIL in monocytic cells whereas tat-expressing lymphoid cells are more resistant to TRAIL cytotoxicity, we next investigated the effect of Tat on the expression/activity of both apical caspase-8 and -10, which play a key role in mediating the initial phases of apoptosis by TRAIL, and c-FLIP. Jurkat lymphoblastoid human T cell lines stably transfected with a plasmid expressing wild-type (HIV-1) tat gene showed normal levels of caspase-8 but significantly decreased levels of caspase-10 at both mRNA and protein levels with respect to Jurkat transfected with the control plasmid or with a mutated (cys22) non-functional tat cDNA. A significant decrease of caspase-10 expression/activity was also observed in transient transfection experiments with plasmid carrying tat cDNA. Moreover, c-FLIP(L) and c-FLIP(S) isoforms were up-regulated in tat-expressing cells at both mRNA and protein level in comparison with control cells. Taken together, these results provide a molecular basis to explain the resistance of tat-expressing Jurkat cells to apoptosis induced by TRAIL and, possibly, to other death-inducing ligands. Copyright 2004 Wiley-Liss, Inc.

Serum antibodies against human intracisternal A-type particle (HIAP) endogenous retrovirus have been found to be associated with various autoimmune pathologies. To evaluate the presence of serum antibody reactivity to HIAP proteins in systemic sclerosis, a Western blot analysis was performed on sera from 42 patients with systemic sclerosis, in comparison with 18 sera from patients with primary biliary cirrhosis and 52 healthy subjects. A positive Western blot was found in 55.5% of serum samples from patients with primary biliary cirrhosis and in 66.0% of patients with systemic sclerosis. None of the 52 healthy subjects showed positive results. Although this difference may be attributable either to an autoimmune response to antigenically related cellular proteins or to a specific antibody response to HIAP proteins expressed as an incidental consequence of attendant pathological processes, the high prevalence of antibodies against HIAP proteins demonstrated in patients with systemic sclerosis may be considered a hallmark of this disease.

Background: The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women. Objective: We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients. Study design: Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors. Results/conclusions: The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients (627±1068 HIV-1 proviral DNA copies per 10 6 PBMC, with a range of 30–6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.

A cross-sectional study was carried out in our tertiary care hospital between January 1998 and December 2001. All 161 consecutive patients naive to nelfinavir and who had received a nelfinavir-based highly active antiretroviral therapy (HAART) of at least 24-week duration were extrapolated from the 802 adult HIV-infected subjects treated with antiretroviral therapy. All cases of virologic failure were considered and viral genotyped. Virologic failure occurred in 80 out of 161 nelfinavir-treated patients, all belonging to the experienced group. On the whole, only 11 patients (7%) developed the D30N substitution, whose 6 was in association with the N88D mutation. Among the 80 failed patients, the M184V mutation was detected in 52 (65%), while only 7 patients showed simultaneously the M184V, T215Y and K103N substitutions. In our HIV-infected population receiving a nelfinavir-based HAART, the D30N mutation has shown a low absolute frequency, while the detection of M184V substitution and the simultaneous occurrence of M184V, T215Y and K103N mutations were related to a more favorable virological response.

The study evaluated the development of drug resistance in a group of HIV-1 patients. After failure to respond to previous therapy with two non-nucleoside reverse transcriptase inhibitors (NNRTIs), as assessed by the presence of a rebound in viral load or a constant high level of HIV plasma viraemia, the patients were treated with a combination of stavudine, lamivudine and efavirenz (EFV). Results showed that viruses carrying primary mutations, usually K103N, T215Y and M41L, presented higher levels of HIV-1 RNA, suggesting an association between a precise mutation pattern and treatment failure.