Embryo morphokinetic score is associated with biomarkers of developmental competence and implantation.

PURPOSE: To study embryo morphokinetics in relation to release in spent media of molecules with possible roles in development and implantation (miR-20a, miR-30c, and sHLA-G). METHODS: Data were obtained from embryos generated in standard IVF and ICSI cycles. The Eeva system was used for embryo assessment, based on early morphokinetic parameters and producing a score (1-5, best-worst) corresponding to higher/medium/lower chances of development to blastocyst. miRNAs - mm miR-20a-5p and miR-30c-5p - and sHLA-G were quantified in 25 µl of spent blastocyst media (SBM) collected before vitrification or transfer. Statistical analyses were performed applying Kolmogorov-Smirnov, Shapiro-Wilk, and Spearman's correlation coefficient tests, where appropriate. RESULTS: SBM were collected from a total of 172 viable blastocysts. Their analysis showed that concentration of miR-20a was progressively lower as Eeva score increased and probability of development to blastocyst decreased (P = 0.016). The opposite trend was observed in the case of miR-30c, i.e., concentration was higher as score increased and chances of development to blastocyst decreased (P = 0.004). Analysis of sHLA-G revealed a negative correlation with Eeva score, i.e., levels were progressively lower as Eeva score increased and probability of development to blastocyst decreased (R = - 0.388, N = 141, P = 0.001). CONCLUSION: Our data suggest that morphokinetic algorithms that predict development to blastocyst stage, in fact, also identify embryos with molecular and cellular profiles more consistent with developmental functions.

Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival.

Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gßγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

Multilevel approach to male fertility by machine learning highlights a hidden link between haematological and spermatogenetic cells.

BACKGROUND: Male infertility represents a complex clinical condition requiring an accurate multilevel assessment, in which machine learning technology, combining large data series in non-linear and highly interactive ways, could be innovatively applied. METHODS: A longitudinal, observational, retrospective, big data study was carried out, applying for the first time the ML in the context of male infertility. A large database including all semen samples collected between 2010 and 2016 was generated, together with blood biochemical examinations, environmental temperature and air pollutants exposure. First, the database was analysed with principal component analysis and multivariable linear regression analyses. Second, classification analyses were performed, in which patients were a priori classified according to semen parameters. Third, machine learning algorithms were applied in a training phase (80% of the entire database) and in a tuning phase (20% of the data set). Finally, conventional statistical analyses were applied considering semen parameters and those other variables extracted during machine learning. RESULTS: The final database included 4239 patients, aggregating semen analyses, blood and environmental parameters. Classification analyses were able to recognize oligozoospermic, teratozoospermic, asthenozoospermic and patients with altered semen parameters (0.58 accuracy, 0.58 sensitivity and 0.57 specificity). Machine learning algorithms detected three haematological variables, that is lymphocytes number, erythrocyte distribution and mean globular volume, significantly related to semen parameters (0.69 accuracy, 0.78 sensitivity and 0.41 specificity). CONCLUSION: This is the first machine learning application to male fertility, detecting potential mathematical algorithms able to describe patients' semen characteristics changes. In this setting, a possible hidden link between testicular and haematopoietic tissues was suggested, according to their similar proliferative properties.

Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: a prospective, blinded, multi-centric study.

In ART, embryo quality evaluation is routinely based on morphological criteria. We previously demonstrated that the mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in culture medium was significantly associated with embryo quality and viability potential. The purpose of this prospective, blinded, multi-centric study was to validate the use of mtDNA/gDNA ratio in Day 3 spent medium as a predictor of human embryo developmental competence. The mtDNA/gDNA ratio was assessed in Day 3 culture media (n=484) of embryos from 143 patients by quantitative PCR. A mixed effect logistic regression model was applied. We found that mtDNA/gDNA ratio in Day 3 culture medium combined with embryo morphology improves the prediction upon blastulation compared to morphology alone (P < 0.0001), independent of patient and cycle characteristics. With regard to routine use in clinics, we evaluated the ability of the novel, combined grading score to improve selection of developmentally competent embryos of a single cohort. Including embryos from 44 patients, the sensibility and specificity of the scoring system based on Day 3 morphological stage were 92% and 13%, respectively. Integration with the culture medium mtDNA/gDNA ratio increased the performance of the method (sensibility: 95%; specificity: 65%). The results of this study suggest the possibility of carrying out a non-invasive evaluation of embryonic mtDNA content through the culture medium. When combined with embryo morphology, it has the potential to help embryologists rank embryos and choose which embryo(s) has the greater development potential, and thus should be transferred on Day 3, among sibling embryos with the same morphological grade.

Incident genital HPV infections and potential impact of HPV vaccines in adult women living with HIV/AIDS.

This study aims to describe and characterize incident HR-HPV infections and associated diseases in HIV-infected women. 805 HIV-infected women enrolled in the VALHIDATE Study were screened and followed-up for HPV by co-testing. Social, behavioral and health data were collected. HPV-DNA positive samples were typed using a commercial kit or RFLP analysis. Conventional Pap-smears were evaluated using the 2001 Bethesda System. The participants with abnormal cytological results were referred for colposcopy. 565 HIV-infected women (median age: 43 years) were analysed, 40.9% had >5 lifetime sexual partners, 77.2% contracted HIV through sexual intercourse, 93% were receiving antiretroviral treatment and 77.3% had undetectable HIV-RNA. The women underwent 1254 follow-ups (median follow-up: 33 months) for 1430.6 PersonYear-Follow-Up. 37.4% of baseline HPV-negative women acquired incident HPV-infections, 69.6% of which were HR-HPVs. HPV-53 was the most common HPV type detected (9.3%). 18.2% of women showed incident or progressive cytological abnormalities (7.8% ASC-US, 9.7% LSIL and 0.6% HSIL) and colposcopy revealed CIN2 (N = 2), CIN1 (N = 2) and VIN3 (N = 1). The preventable fraction of incident infections was 11.3%, 16.7%, and 35.2% for the 2v-4v-9v-HPV vaccines respectively (χ2 p < 0.0001). The overall burden of incident lesions attributable to the vaccine types were 9.1% for 2v-, 14.5% for 4v- and 30.9% for 9v-vaccine. High HPV incidence rates and high percentages of multiple HR-HPV infections were observed in a cohort of HIV-infected women receiving effective antiretroviral treatment. Primary prevention strategies based on the new 9v-HPV vaccine may help to prevent incident infections and disease progression in this cohort of women.

Biochemical pregnancy loss after frozen embryo transfer seems independent of embryo developmental stage and chromosomal status.

RESEARCH QUESTION: Biochemical pregnancy loss (BPL), defined as serum beta-human chorionic gonadotropin levels ≥50 IU/l in at least two pregnancy tests, not associated with any ultrasonographical evidence of pregnancy, is often attributed to chromosomal abnormalities; however, no hard evidence exists to support this hypothesis. Are any IVF cycle parameters associated with the occurrence of a BPL? DESIGN: Retrospective study aimed at evaluating the effect of embryo developmental stage at transfer and chromosomal assessment on the BPL rate in IVF after frozen embryo transfer (FET). Specifically, 641 FET of 1179 cleavage stage untested embryos (Group A), 1021 FET of 1259 untested blastocyst stage embryos (Group B), and 789 blastocyst stage FET of 803 euploid embryos (Group C) were performed in a 6-year period. Only FET were evaluated to avoid a potential effect of ovarian stimulation on endometrial receptivity. RESULTS: The BPL rates were similar (n = 30/217, 13.8% in Group A; n = 37/412, 9.0% in Group B; n = 42/433, 9.7% in Group C). Neither embryo developmental stage at FET nor chromosomal assessment showed a correlation with BPL. Furthermore, logistic regression analyses did not show any association between BPL and patient, cycle and/or transfer characteristics. CONCLUSIONS: BPL seems independent of the embryo's developmental stage, the use of trophectoderm biopsy and the chromosomal constitution at FET. Similar BPL rates after transferring euploid blastocysts compared with both untested cleavage and blastocyst stage embryos suggest investigating the role of endometrial and other embryonic factors putatively involved in the process of implantation.

Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation.

STUDY QUESTION: Are trophectoderm biopsy or other pre-vitrification features or laboratory practices associated with differences in blastocyst post-warming behavior (degeneration, re-expansion and live birth after single embryo transfer (SET))? SUMMARY ANSWER: Blastocyst morphology, day of full development and artificial shrinkage (either laser-assisted or biopsy-induced) are the pre-vitrification parameters/practices most strongly associated with post-warming behavior and implantation potential while there was no association with trophectoderm biopsy. WHAT IS KNOWN ALREADY: Since the introduction of vitrification, the adoption of cycle segmentation, freeze-all and SET policies, as well of trophectoderm biopsy-based aneuploidy testing (i.e. pre-implantation genetic testing for aneuploidies (PGT-A)), the number of blastocysts vitrified worldwide has increased greatly. Previous studies already defined generally high blastocyst cryo-survival rates after vitrification-warming (>95%), along with a positive correlation between blastocyst re-expansion and morphology with implantation. Additionally, artificial shrinkage has been outlined as a potentially beneficial procedure, while the association between embryo cryo-survival and trophectoderm biopsy is still unclear. STUDY DESIGN, SIZE, DURATION: Cohort study conducted at two IVF centers (1 and 2). A total of 2129 consecutive SETs using vitrified-warmed blastocysts in either non-PGT or PGT-A cycles between June 2016 and August 2017 were included. A freeze-all strategy was in place and three main pre-vitrification practices were used: (i) no biopsy and no artificial shrinkage (Clinic 1); (ii) trophectoderm biopsy and vitrification of collapsed blastocyst within 30 min (Clinics 1 and 2); and (iii) no biopsy but laser-assisted artificial shrinkage (Clinic 2). The primary outcome was the blastocyst degeneration rate. Overall, 2108 surviving blastocysts were graded at 1.5 h after warming for degeneration (absent or partial) and re-expansion (full, partial or absent) grades and post-warming morphological quality. Logistic regression analyses were conducted to assess the association of any pre-vitrification feature with blastocyst post-warming behavior. The logistic regressions conducted upon live birth after either untested or euploid SET also included the post-warming characteristics. PARTICIPANTS/MATERIALS, SETTING, METHODS: Center 1 is a private IVF facility, while center 2 is the IVF facility of a University hospital. In non-PGT cycles, ICSI with blastocyst culture up to full-expansion and vitrification were performed. At center 1 the untested blastocysts were vitrified when still expanded, while at center 2 they underwent laser-assisted artificial shrinkage. In PGT-A cycles, in both clinics, trophectoderm biopsy (which involves laser-assisted shrinkage) was done without previous zona-opening on Day 3, and vitrification was performed within 30 min whilst the blastocyst remained collapsed. A qPCR-based chromosome analysis was conducted. Only SETs were performed (euploid-SET in case of PGT-A). Any cycle-, laboratory- and embryo-based feature which could impact blastocyst post-warming behavior was included in the analyses as putative confounder. MAIN RESULTS AND THE ROLE OF CHANCE: The overall degeneration rate was 1% (N = 21/2129). The results were consistent among different vitrification/warming operators or kits used, as well as any other IVF laboratory-related parameter. Blastocyst artificial shrinkage (either laser-assisted or biopsy-induced) involved a lower risk of degeneration after warming (odds ratio (OR) [95% CI] = 0.26 [0.09-0.79]). Conversely, both poor morphological quality pre-vitrification and taking 7 days to reach full blastulation resulted in a significantly higher risk (OR [95% CI] = 11.67 [3.42-39.83] and 4.43 [1.10-20.55], respectively). Importantly, trophectoderm biopsy did not show any association with blastocyst cryo-survival/degeneration. Overall 2.5% (N = 53/2108) blastocysts failed to re-expand post-warming. The only parameters significantly associated with no blastocyst re-expansion post-warming were average (OR [95% CI] = 4.96 [2.20-11.21]) or poor (OR [95% CI] = 19.54 [8.39-45.50]) morphological quality and taking 7 days to reach full blastulation (OR [95% CI] = 3.19 [1.23-8.29]), as well as prevention of spontaneous hatching pre-vitrification (OR [95% CI] = 0.10 [0.01-0.85]). The post-warming features of the survived blastocyst (i.e. degeneration and re-expansion scores and morphological quality) showed no significant association with vitrified blastocyst competence (i.e. live birth) when corrected for pre-vitrification ones (i.e. morphological quality, day of full development and, for untested SET, maternal age at oocyte retrieval). Of note, poor-quality blastocysts pre-vitrification showed a high overall cryo-survival rate post-warming 92.8% (N = 116/125), but the live birth rates were only 2.1% (N = 1/48) and 7.3% (N = 5/68) after untested and euploid SET, respectively. LIMITATIONS, REASONS FOR CAUTION: This study is not randomized and the populations of patients undergoing either non-PGT or PGT-A cycles were different. Centers 1 and 2 adopted different pre-vitrification practices for non-biopsied blastocysts, according to their own laboratory policy. To this regard, multivariate logistic regression analyses were conducted for all outcomes under investigation. WIDER IMPLICATIONS OF THE FINDINGS: Pre-vitrification features may be used to assist selection of competent embryos, moreover, these results allay concern that trophectoderm biopsy might be associated with impaired blastocyst quality or competence after vitrification/warming. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

Abnormally fertilized oocytes can result in healthy live births: improved genetic technologies for preimplantation genetic testing can be used to rescue viable embryos in in vitro fertilization cycles.

OBJECTIVE: To test whether abnormally fertilized oocyte (AFO)-derived blastocysts are diploid and can be rescued for clinical use. DESIGN: Longitudinal-cohort study from January 2015 to September 2016 involving IVF cycles with preimplantation genetic testing for aneuploidy (PGT-A). Ploidy assessment was incorporated whenever a blastocyst from a monopronuclear (1PN) or tripronuclear zygote (2PN + 1 smaller PN; 2.1 PN) was obtained. SETTING: Private IVF clinics and genetics laboratories. PATIENT(S): A total of 556 women undergoing 719 PGT-A cycles. INTERVENTION(S): Conventional chromosome analysis was performed on trophectoderm biopsies by quantitative polymerase chain reaction. For AFO-derived blastocysts, ploidy assessment was performed on the same biopsy with the use of allele ratios for hetorozygous SNPs analyzed by means of next-generation sequencing (1:1 = diploid; 2:1 = triploid; loss of heterozygosity = haploid). Balanced-diploid 1PN- and 2.1PN-derived blastocysts were transferred in the absence of normally fertilized transferable embryos. MAIN OUTCOME MEASURE(S): Ploidy constitution and clinical value of AFO-derived blastocysts in IVF PGT-A cycles. RESULT(S): Of the 5,026 metaphase II oocytes injected, 5.2% and 0.7% showed 1PN and 2.1PN, respectively. AFOs showed compromised embryo development (P<.01). Twenty-seven AFO-derived blastocysts were analyzed for ploidy constitution. The 1PN-derived blastocysts were mostly diploid (n = 9/13; 69.2%), a few were haploid (n = 3/13; 23.1%), and one was triploid (n = 1/13; 7.7%). The 2.1PN-derived blastocysts were also mostly diploid (n = 12/14; 85.7%), and the remainder were triploid. Twenty-six PGT-A cycles resulted in one or more AFO-derived blastocysts (n = 26/719; 3.6%). Overall, eight additional balanced-diploid transferable embryos were obtained from AFOs. In three cycles, the only balanced-diploid blastocyst produced was from an AFO (n = 3/719; 0.4%). Three AFO-derived live births were achieved: one from a 1PN zygote and two from 2.1PN zygotes. CONCLUSION(S): Enhanced PGT-A technologies incorporating reliable ploidy assessment provide an effective tool to rescue AFO-derived blastocysts for clinical use.

Pre-implantation genetic testing in ART: who will benefit and what is the evidence?

Pre-implantation genetic diagnosis for aneuploidy testing (PGD-A) is a tool to identify euploid embryos during IVF. The suggested populations of patients that can benefit from it are infertile women of advanced maternal age, with a history of recurrent miscarriages and/or IVF failures. However, a general consensus has not yet been reached.After the clinical failure of its first version based on cleavage stage biopsy and 9 chromosome-FISH analysis, PGD-A is currently performed by 24 chromosome screening techniques on trophectoderm (TE) biopsies. This approach has been clearly demonstrated to involve a higher clinical efficiency with respect to the standard care, in terms of sustained pregnancy rate per transfer and lower miscarriage rate. However, data about PGD-A efficacy calculated on a per intention-to-treat basis, as well as an analysis of its cost-effectiveness, are still missing.TE biopsy is a safe and extensively validated approach with low biological and technical margin of error. Firstly, the prevalence of mosaic diploid/aneuploid blastocysts is estimated to be between 0 and 16 %, thus largely tolerable. Secondly, all the comprehensive chromosome screening (CCS) technologies adapted to, or designed to conduct PGD-A are highly concordant, and qPCR in particular has been proven to show the lowest false positive error rate (0.5 %) and a clinically recognizable error rate per blastocyst of just 0.21 %.In conclusion, there is a sufficient body of evidence to support the clinical application of CCS-based PGD-A on TE biopsies. The main limiting factor is the need for a high-standard laboratory to conduct blastocyst culture, biopsy and vitrification without impacting embryo viability.

A quantitative approach to blastocyst quality evaluation: morphometric analysis and related IVF outcomes.

PURPOSE: To quantify blastocyst morphologic parameters with a feasible and standardized tool, investigating their predictive value on implantation outcome. METHOD: The study retrospectively analyzes 124 blastocysts from 75 patients. Quantitative measurements of blastocyst expansion, inner cell mass and trophoectoderm were taken using digital image analysis software. RESULT(S): Blastocysts areas were found to be ranging from 11626.2 up to 35076.4 µm(2). The area of an early blastocyst is A ≤ 18500 µm(2) with a mean diameter d = 140 ± 9 µm, and the area of an expanded blastocyst is A ≥ 24000 with d = 190 ± 9 µm. While blastocyst mean area was not related to implantation rate, more expanded blastocysts displayed a significantly higher implantation rate. Trophoectoderm cell number is a predictor of positive outcome: since a higher of cells (25.6 ± 11.3 vs 16.3 ± 12.8) `forming a tightly knit epithelium is prognostic of implantation potential. Conversely, inner cell mass size is significantly related to implantation only in expanded blastocysts (3122.7 ± 739.0 vs. 2978.1 ± 366.0 µm(2)). CONCLUSION(S): Evaluation of blastocyst morphology with a digital image system could be a valuable tool to standardize blastocyst grading based on quantitative parameters. Therefore, digital analysis may be helpful in identifying the best blastocyst to transfer.

Trends in hospital admissions at a Department for Infectious Diseases in Italy from 1995 to 2011 and implications for health policies.

BACKGROUND: Interactions among several environmental, behavioral, social, and biological variables contribute to the epidemiology of infectious diseases (IDs) and have an impact on the healthcare system and hospitalizations. We evaluated trends in ID hospitalizations at our Department for Infectious Diseases in the last two decades to aid decision-makers in defining appropriate healthcare strategies. METHODS: The discharge diagnoses of all patients admitted to the ID Department of L Sacco University Hospital between 1995 and 2011 were classified by the International Classification of Diseases (ICD-9) grouped in Major Diagnostic Categories (MDC). Linear regression was used to determine the trends in hospitalizations for each MDC. Estimates of the average annual change were based on the slope of the regression line. RESULTS: A sharp decline in HIV/AIDS cases (-22.5 +/-6.0 cases per calendar year), and an increase in admissions for respiratory, cardiovascular, renal and musculoskeletal infections were recorded. The mean age of the patients increased by 1.2 years (+/-0.049) for each calendar year of observation (linear trend, p < 0.0001), increasing from 37.02 +/-11.91 years in 1995 to 56.02 +/-19.62 years in 2011 (p < 0.0001). The mean number of comorbidities per patient increased significantly over time (Mann-Whitney U test, p = 0.0153). From 1998/1999 to 2010/2011 the hospital length of stay (LOS) increased for cardiovascular, digestive system, musculoskeletal, and skin/subcutaneous infections, and infectious and parasitic diseases (p < 0.01). The rate of hospital stay over threshold (HSOT) increased in the last 5 years by 1.12% for every 10-year age group. CONCLUSIONS: Older age, a higher number of comorbidities, a longer hospital LOS for certain conditions, and a higher rate of HSOT characterize the patients admitted to this ID department in recent years. Despite progress in treatment and management, infectious diseases continue to be a major threat to human health. The current challenge for ID departments is the treatment of complex cases, often associated with chronic diseases in elderly patients. Continuous monitoring at a local and national level will allow early identification of changes in the epidemiological patterns of IDs and provide information for healthcare system planning.

Fungal endocarditis observed over an 8-year period and a review of the literature.

BACKGROUND: Fungal endocarditis (FE) is a "modern" disease that is considered an emerging cause of infective endocarditis (IE). The most frequently identified fungal pathogens are Candida spp., which are responsible for up to two-thirds of all cases; the remaining cases are due to Aspergillus spp., Histoplasma capsulatum or, more rarely, other yeasts and moulds. OBJECTIVES: To describe the prevalence, clinical characteristics and outcome of FE diagnosed in a single tertiary centre and review the literature concerning FE. DESIGN AND SETTING: An 8-year retrospective review of the case records of patients attending a single Italian University Centre and diagnosed as having definite or probable IE as defined by the modified Duke criteria. RESULTS: Six patients were identified from 229 episodes of IE: five cases involved a prosthetic valve, and one a native valve of an intravenous drug user. Five cases were caused by Candida spp. (two by C. albicans, one each by C. lusitaniae, C. dubliniensis and C. glabrata) and one by Aspergillus flavus. Three patients were treated by means of surgery plus antifungal therapy; two received antifungal therapy alone. Three patients survived, but only the patient with Aspergillus endocarditis was followed up for a long time. CONCLUSIONS: FE is difficult to diagnose but generally associated with healthcare infections. The optimal treatment is poorly characterised, and international collaborative studies are urgently needed to evaluate newer antifungal agents.

Risk of cervical HPV infection and prevalence of vaccine-type and other high-risk HPV types among sexually active teens and young women (13-26 years) enrolled in the VALHIDATE study.

HPV vaccination is expected to reduce the incidence of cervical cancer. The greatest and the earliest health gains will be ensured by high vaccine coverage among all susceptible people. The high costs and the risk of a reduced cost/effectiveness ratio in sexually active girls still represent the main obstacles for a more widespread use of HPV vaccination in many countries. Data on the rate, risk factors, and HPV types in sexually active women could provide information for the evaluation of vaccination policies extended to broader age cohorts. Sexually active women aged 13-26 years enrolled in an Italian cohort study were screened for cervical HPV infections; HPV-DNA positive samples were genotyped by InnoLipa HPV Genotyping Extra or by RFLP genotype analysis.: Among the 796 women meeting the inclusion criteria, 10.80% (95% CI 8.65-12.96) were HPV-DNA infected. Age>18 years, lifetime sexual partners>1, and history of STIs were associated to higher risk of HPV infection in the multivariable models adjusted for age, lifetime sexual partners, and time of sexual exposure. The global prevalence of the four HPV vaccine-types was 3.02% (95% CI 1.83-4.20) and the cumulative probability of infection from at least one vaccine-type was 12.82% in 26-years-old women and 0.78% in 18-years-old women.: Our data confirm most of the previously reported findings on the risk factors for HPV infections. The low prevalence of the HPV vaccine-types found may be useful for the evaluation of the cost/efficacy and the cost/effectiveness of broader immunization programs beyond the 12-years-old cohort.

Profile of infective endocarditis observed from 2003 - 2010 in a single center in Italy.

BACKGROUND: This study aimed to provide a contemporary picture of the epidemiologic, clinical, microbiologic characteristics and in-hospital outcome of infective endocarditis (IE) observed in a single center in Italy. METHODS: We performed a retrospective study of patients with definite or probable IE observed at the "L. Sacco" Hospital in Milan, Italy, from January 1, 2003 through December 31, 2010. RESULTS: 189 episodes of IE in 166 patients were included. The mean number of incident IE in the study period was of 1.27 (range 0.59-1.76) cases per 1000 patients admitted. The median age of the cohort was 57 (interquartile range, 43-72) years, 63% were male and 62.5% had native valve IE. Twenty-six percent were active intravenous drug users (IVDU), 29% had a health care-associated IE and 5% chronic rheumatic disease. Twenty-nine percent of the cases occurred in patients affected by chronic liver disease and 19% in HIV positive subjects. Staphylococcus aureus was the most common pathogen (30%), followed by streptococci. The mitral (34%) and aortic (31%) valves were involved most frequently. The following complications were common: stroke (19%), non-stroke embolizations (25%), heart failure (26%) and intracardiac abscess (9%). Surgical treatment was frequently employed (52%) but in hospital mortality remained high (17%). Health care-associated IE and complications were independently associated with an increased risk of in-hospital death, while surgery was associated with decreased mortality. CONCLUSION: S. aureus emerged as the leading causative organism of IE in a University hospital in northern Italy. Our study confirmed the high in-hospital mortality of IE, particularly if health care associated, and the protective role of surgery.

Linezolid plasma concentrations and occurrence of drug-related haematological toxicity in patients with gram-positive infections.

Retrospective studies have documented a significant association between linezolid (LNZ) plasma concentrations and drug-related haematological toxicity. However, the safe upper threshold level for LNZ plasma trough concentrations (Cmin values) has not been defined with certainty. A prospective observational study was performed aimed at comparing LNZ Cmin values in patients developing drug-related side effects with those measured in patients not experiencing LNZ toxicity. LNZ Cmin values were measured from the first week after starting therapy and were repeated periodically up to the end of treatment. Fifty patients, for a total of 210 LNZ Cmin evaluations, were considered. All patients (n=9) who developed drug-related haematological toxicity also had significantly higher plasma LNZ Cmin values during the first week of therapy (9.0±6.4 mg/L vs. 4.9±3.7 mg/L; P<0.01) and thereafter (9.3±5.4 mg/L vs. 4.4±3.4 mg/L; P<0.01). The significant association between LNZ plasma concentrations and haematological toxicity was also confirmed by multivariate logistic regression analysis including age, serum creatinine and concomitant medications as independent variables. A causal relationship between LNZ concentrations and the risk of developing drug-related haematological toxicity was observed. Accordingly, application of therapeutic drug monitoring may improve the safety outcome of patients receiving LNZ therapy.

High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs.

Cost-effectiveness analysis of HIV treatment in the clinical practice of a public hospital in northern Italy.

INTRODUCTION: We carried out an economic analysis to assess the cost-effectiveness of highly active antiretroviral therapy (HAART) regimens in Italy for the management of human immunodeficiency virus (HIV)-infected patients according to clinical practice in the Infectious Diseases Department of "L. Sacco" Hospital, Milan, Italy. PATIENTS AND METHODS: The incremental cost-effectiveness analysis was completed by means of a Markov model. Through a decision-analytic approach, this enabled us to compare the studied antiretroviral regimens. The model considered a population of adult HIV subjects who received HAART therapy for the first time according to clinical practice in the Infectious Diseases Department of "L. Sacco" Hospital, Milan. Data were investigated from the standpoint of the Lombardy Regional Health Service. We considered the following outcome measures: quality-adjusted life-years (QALYs), and direct health costs calculated for the years 2008 and 2009. The time horizon adopted in the model was 2 years. RESULTS: The model revealed that, in terms of cost per gained QALY, the tenofovir disoproxil fumarate + emtricitabine + efavirenz (TDF+FTC+EFV) once-a-day treatment strategy seems to be the most cost-effective therapeutic choice (€34,965); the incremental cost-effectiveness ratio (ICER) values for the remaining strategies ranged from €53,000 to around €62,000 per QALY. The validity of the base case scenario was then confirmed by means of a sensitivity analysis on the main variables. CONCLUSION: The TDF+FTC+EFV treatment strategy (TDF/FTC+EFV fixed-dose combination then switched to single-tablet regimen [STR]) in this setting is the most cost-effective treatment strategy compared with the other therapeutic regimens. The ICER value for the TDF+FTC+EFV once-a-day then switched to STR treatment was lower than the internationally generally accepted threshold value of €50,000. The developed model is a tool for policy makers and health care professionals for creating short- and long-term cost projections, with the aim of assessing their impact on the available budgets for HIV patients.

Rationale and design of a multicenter prospective cohort study for the eVALuation and monitoring of HPV infections and relATEd cervical diseases in high-risk women (VALHIDATE study).

BACKGROUND: Pap screening, an effective method for cervical cancer prevention, is now supported by molecular human papillomavirus (HPV) testing. Recently commercialised preventive vaccines also provide new tools for the primary prevention of cervical cancer. To determine appropriate prevention strategies, the Health General Direction, Lombardy Region, funded a project that aims to characterize and monitor HPV infections and related cervical diseases in high-risk women. METHODS/DESIGN: VALHIDATE is a 5-year multicentre open prospective cohort study. It will recruit 7000 consenting women aged 13-65 years to provide information about the local biomolecular epidemiology of HPV infection and cervical diseases in high-risk women recruited from nine clinical centres and one faith-based organisation. The study will estimate the overall and type-specific prevalence of HPV infection and cervical abnormalities. It also aims to compare standard Pap screening with biomolecular screening, and to assist in the design of targeted regional prevention programs directed specifically at high-risk groups. Three groups of high-risk women: 1000 HIV-infected women (aged 26-65 years), 1000 recent migrant women (aged 26-65 years) and 3000 young women (aged 13-26 years) and 1 control group: 2000 women (aged 26-45 years) attending a spontaneous screening program, will be recruited. Sample sizes will be revised after the first year. Adult participants will undergo conventional cervical cytology, HPV DNA screening and genotyping. Paediatric participants will undergo HPV DNA testing and genotyping of urine samples. HPV DNA, cytological abnormalities and HPV types will be analysed according to demographic, epidemiological, behavioural, and clinical data collected in an electronic case report form. Overall and stratified prevalences will be estimated to analyse the associations between HPV infection and selected characteristics. Logistic regression models will be used to estimate crude and adjusted odds ratios. Cox proportional hazard models will be used to estimate hazard ratios over time and between groups. DISCUSSION/MAIN EXPECTED RESULTS: This study will provide substantial insight into HPV infections and related cervical diseases in high-risk groups and will help determine appropriate regional cervical cancer prevention strategies.

Diagnosis and therapy for hospitalized imported malaria in adults in Italy.

BACKGROUND: The diagnosis and treatment of malaria in non-endemic countries presents a continuing challenge. METHODS: Medical records were reviewed for 291 patients hospitalized with microscopically confirmed malaria diagnosed consecutively in two infectious diseases wards in Milano, Italy, between 1998 and 2007. RESULTS: One hundred eighty-six (64%) were male; median age was 35 y (range 16-72 y). Of the 291 patients, 204 (70.1%) were non-immune travelers and 87 (29.9%) were considered semi-immune. In 228 patients (78.3%), Plasmodium falciparum was identified as the only causative malarial parasite. In 48 (16.5%), 9 (3.1%), and 1 (0.3%) cases, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae were diagnosed, respectively. Five mixed infections were observed (1.7%). Of the 233 falciparum cases (including mixed infections), 222 (95.3%) were acquired in sub-Saharan Africa. Fifty-four percent of P vivax infection were acquired in the Indian subcontinent and Southeast Asia. Chemoprophylaxis was used by 23.6% (61/258) subjects with only 32 fully compliant with the recommended regimen. At admission, fever, chills, and headache were present in 95.5, 59.5, and 55.3% of cases, respectively. Elevated serum lactate dehydrogenase levels (95%) and thrombocytopenia (82%) were the most frequently detected laboratory abnormalities. Thirty-five patients (15%) with P falciparum malaria presented with severe malaria according to the WHO criteria; in 19 patients (54.3%) more than one criteria was present. All patients recovered uneventfully. Inappropriate anti-malarial treatment occurred in 25 patients (8.6%) and were recorded more frequently among patients with a diagnosis of P vivax malaria (29.1%) as opposed to those affected by P falciparum (3.9%). CONCLUSIONS: In our study more than two thirds of imported malaria cases were due to P falciparum with an excess of cases diagnosed in immigrants starting from the year 2000. Despite many available guidelines inappropriate initial malaria treatment is relatively frequent even when patients are managed in an infectious diseases ward.