Detection of hepatitis C virus in an exhumed body identified the origin of a nosocomial transmission that caused multiple fatal diseases.

BACKGROUND: Medico-legal conflicts arise when it is difficult to prove the cause of nosocomial infections. AIM: To report an outbreak of patient-to-patient transmission of hepatitis C virus (HCV) through the repeated use of a multi-dose saline flask during the rinsing of central venous catheters. METHODS: Blood samples were taken from each patient for the comparative analysis of their HCV RNA strains. No samples were available for one patient who died before the investigation started. Despite the known lability of HCV RNA, the body was exhumed four months after burial and postmortem samples were collected. HCV RNA was extracted successfully from liver and spleen samples. Genotyping of all the HCV strains was performed by sequence analysis of the 5'NC untranslated region, the E1 core conserved region and the E1/E2 hypervariable region. FINDINGS: Forensic investigators retraced the route used by two ward nurses, when saline catheter flushes were given to 14 patients with each nurse administering to seven patients. The comparative phylogenetic analysis of all case strains identified the deceased patient as the source of contamination to five patients. CONCLUSIONS: This study highlights the value of sequence analysis as a tool for solving medico-legal conflicts. The High Court of Justice found that a health worker's re-use of a contaminated needle resulted in the nosocomial transmission of HCV.

Colonization and infection due to carbapenemase-producing Enterobacteriaceae in liver and lung transplant recipients and donor-derived transmission: a prospective cohort study conducted in Italy.

OBJECTIVES: A prospective cohort study was conducted in Italy in order to describe the microbiologic aspects of colonization/infection by carbapenemase-producing Enterobacteriaceae (CPE) in donors and recipients of lung and liver transplants and the possible CPE transmission from donors to recipients. METHODS: Between 15 January 2014 and 14 January 2015, all recipients of solid organ transplants (SOT) at ten lung and eight liver transplantation centres and the corresponding donors were enrolled. Screening cultures to detect CPE were performed in donors, and screening and clinical cultures in recipients with a 28-day microbiologic follow-up after receipt of SOT. Detection of carbapenemase genes by PCR, genotyping by multilocus sequence typing, and pulsed-field gel electrophoresis and whole-genome sequencing were performed. RESULTS: Of 588 screened donors, 3.4% were colonized with CPE. Of the liver first transplant recipients (n = 521), 2.5% were colonized before receipt of SOT and 5% acquired CPE during follow-up. CPE colonization was higher in lung first transplant recipients (n = 111, 2.7% before SOT and 14.4% after SOT). CPE infections occurred in 1.9% and 5.3% of liver or lung recipients, respectively. CPE isolates were mostly Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae belonging to CG258. Three events of donor-recipient CPE transmission, confirmed by whole-genome sequencing and/or pulsed-field gel electrophoresis, occurred in lung recipients: two involving K. pneumoniae sequence type 512 and one Verona integron-encoded metallo-ß-lactamase (VIM)-producing Enterobacter aerogenes. CONCLUSIONS: This study showed a low risk of donor-recipient CPE transmission, indicating that donor CPE colonization does not necessarily represent a contraindication for donation unless colonization regards the organ to be transplanted. Donor and recipient screening remains essential to prevent CPE transmission and cross-infection in transplantation centres.

Modeling and improving Ethiopian pasture systems.

The production of pasture in Ethiopia was simulated by means of a dynamic model. Most of the country is characterized by a tropical monsoon climate with mild temperatures and precipitation mainly concentrated in the June-September period (main rainy season). The production model is driven by solar radiation and takes into account limitations due to relocation, maintenance respiration, conversion to final dry matter, temperature, water stress, and nutrients availability. The model also considers the senescence of grassland which strongly limits the nutritional value of grasses for livestock. The simulation for the 1982-2009 period, performed on gridded daily time series of rainfall and maximum and minimum temperature with a resolution of 0.5°, provided results comparable with values reported in literature. Yearly mean yield in Ethiopia ranged between 1.8 metric ton per hectare (t ha-1) (2002) and 2.6 t ha-1 (1989) of dry matter with values above 2.5 t ha-1 attained in 1983, 1985, 1989, and 2008. The Ethiopian territory has been subdivided in 1494 cells and a frequency distribution of the per-cell yearly mean pasture production has been obtained. This distribution ranges from 0 to 7 t ha-1 and it shows a right skewed distribution and a modal class between 1.5-2 t ha-1. Simulation carried out on long time series for this peculiar tropical environment give rise to as lot of results relevant by the agroecological point of view on space variability of pasture production, main limiting factors (solar radiation, precipitation, temperature), and relevant meteo-climatic cycles affecting pasture production (seasonal and inter yearly variability, ENSO). These results are useful to establish an agro-ecological zoning of the Ethiopian territory.

Strong and persistent correlation between baseline and follow-up HIV-DNA levels and residual viremia in a population of naïve patients with more than 4 years of effective antiretroviral therapy.

In a longitudinal study on 181 naïve patients who responded to therapy (mean follow-up 4 years), high baseline human immunodeficiency virus (HIV)-RNA values correlated with high levels of cellular HIV-DNA at all time points (p < 0.0001, p 0.045, p 0.0055, and p 0.0025, respectively) and negatively correlated with undetectable residual viremia (URV; <2.5 copies/mL) at T1, T2, and T3 (p 0.026, p 0.0149, and p 0.0002, respectively). Baseline high HIV-DNA levels predicted the persistence of high values (p 0.0001) and negatively correlated with URV (p 0.0254, p 0.0481, and p 0.0085). These results suggest that baseline viral load, cellular HIV-DNA, and URV were strongly correlated over long-term follow-up of antiretroviral therapy responders.

Climate change in Europe and effects on thermal resources for crops.

Atmospheric variables play a fundamental role in driving man-managed ecosystems and more specifically in agro-ecosystems, determining the quantity and quality of crop production. On the other hand, climate variability can be seen as the superimposition of gradual and abrupt changes. This paper is focused on European surface air temperature in the period 1951-2010. Analysis of this dataset identified breakpoints that define two homogeneous sub-periods: 1951-1987 and 1988-2010. Thermal resources for crops were analyzed adopting a "normal heat hours" approach. Computation highlighted a general increase in thermal resources in the European continent for crop groups II and III (C3 and C4 plants adapted to high or moderate temperatures), while a decline of thermal resources for crop group I (cold adapted C3) was highlighted in the Mediterranean area. The climate variability justifies a change in the potential latitudinal limits of different groups of crops, representing a fundamental step for crop adaptation to climate change.

HIV coreceptor tropism in paired plasma, peripheral blood mononuclear cell, and cerebrospinal fluid isolates from antiretroviral-naïve subjects.

A survey of HIV coreceptor usage in cerebrospinal fluid (CSF) samples, peripheral blood mononuclear cells (PBMCs), and plasma samples from naïve seropositive patients was conducted. One hundred patients were enrolled in this study. Of the 100 patients, 36 had a primary or recent infection (P-RI), 31 had an early chronic infection (>350 CD4 cells) (ECI), and 33 had a late chronic infection (LCI). All 3 compartments were sampled in a subset of 33 participants, while the remaining 67 patients provided plasma samples and PBMCs only. Seventy-seven patients harbored the R5 virus in plasma samples and had a significantly higher median and percentage of CD4(+) T cells than patients with X4 virus (437 and 281 cells/µl, respectively; P = 0.0086; 20.6% and 18.6%, respectively). The X4 strain was detected more frequently in patients with LCI than in patients with P-RI or ECI (39.3%, 19.4%, and 9.6%, respectively; P = 0.0063). PBMC and plasma tropism was concordant in 90 patients, and 73 had the R5 strain. Among patients with discordant results, 4 had the R5 virus in their plasma and the X4 virus in PBMCs; 6 showed the opposite profile. Plasma, PBMC, and CSF tropism determinations were concordant in 26/33 patients (21 patients had R5, and 5 had X4). The tropism was discordant in 5/33 patients, with the X4 virus in plasma and R5 in CSF; the HIV tropism in PBMCs was X4 in 3 patients. The remaining 2/33 patients had the R5 virus in plasma and PBMCs and the X4 virus in CSF; one of these patients had a P-RI. The discordant tropism in CSF and blood may have implications for chemokine (C-C motif) receptor 5 (CCR5) antagonist use in patients with limited response to antiretroviral therapy (ART) or in responding patients evaluated for simplification of treatment.

Molecular epidemiology of Enterococcus faecium isolates from an Italian hospital.

BACKGROUND: Outbreaks of vancomycin-resistant Enterococcus faecium (VRE) strains is an emerging problem worldwide. Even if still relatively uncommon in European hospitals, infections caused by VRE have also been increasing recently in this continent. METHODS: In this study, we characterized 50 consecutive VRE and 23 vancomycin-sensitive E. faecium (VSE) isolates collected in an Italian hospital. The presence of the esp gene and that of genes encoding resistance to glycopeptides was investigated by polymerase chain reaction (PCR). All of the isolates were typed by multi-locus sequence typing (MLST), and a selection of them also by pulsed-field gel electrophoresis (PFGE). RESULTS: We found that all of the VRE and 18 (78%) of the VSE strains belonged to the single clonal complex-17 (CC17). The most represented sequence type (ST) was ST78 (34% of the isolates). When further analyzed by PFGE, ST78 isolates were subdivided into five pulsotypes, four of them closely related. The strong association between the esp gene and CC17 was confirmed. Interestingly, such an association was higher among vancomycin-resistant isolates. Most of the esp-positive isolates (34/46, 74%) encoded Esp4, a rare variant of this protein characterized by the absence of A repeats. CONCLUSIONS: Our findings underscore the role of the CC17 lineage in the nosocomial spread of VRE and VSE, and its rapid local evolution, underscoring the need for programs designed to provide early detection in order to prevent its spreading among the nosocomial population.

Switching of inferred tropism caused by HIV during interruption of antiretroviral therapy.

After interruption of highly active antiretroviral therapy, 15 out of 53 patients with the X4 HIV strain had a significantly larger decrease in CD4(+) T cell count (P = 0.001) and shorter length of treatment interruption (P = 0.02) than patients with the R5 strain. At treatment resumption, HIV inferred tropism switched from the X4 strain to the R5 variant in 9 patients (60%). These patients had a prolonged length of treatment interruption compared to that of those who still carried the X4 strain.

Clustering and risk factors of methicillin-resistant Staphylococcus aureus carriage in two Italian long-term care facilities.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a well-recognized agent of health care-associated infections in long-term care facilities, but few data about the circulation of MRSA in this setting in Italy are available. The aim of the study is to determine the prevalence and risk factors for MRSA carriage in nursing home residents in Vicenza (northeastern Italy). PATIENTS AND METHODS: A point prevalence survey was conducted in two long-term care facilities (subdivided into 15 wards) from 12 June 2006 to 6 July 2006. Anterior nasal swabs were obtained from residents and laboratory screening for MRSA was performed; full antibiotic susceptibility was assessed in MRSA isolates. Macrorestriction analysis of chromosomal DNA was carried out by pulsed field gel electrophoresis (PFGE). For each subject, demographic data, length of stay, dependency, cognitive function, presence of medical devices, comorbidities, current and previous antibiotic treatment, previous hospital admission and presence of infection were assessed on the day of sample collection. Factors that were found to be significantly associated with MRSA carriage at univariate analysis were introduced into multilevel logistic regression models in order to estimate the odds ratios (OR) with 95% confidence intervals (CI) for the risk of MRSA colonization, taking into account the clustering of patients within wards. RESULTS: Nasal swabs were obtained in 551 subjects; overall 43 MRSA carriers were detected (7.8%; CI = 5.7-10.4%). The rate of nasal carriers was very similar in the two institutions, and varied from 0% (0/36) to 18% (7/39) between wards. Only two out of 15 wards were found to have no MRSA carriers; overall, three pairs of colonized roommates were detected. Upon multilevel logistic regression, the risk of MRSA carriage was increased in patients with cancer (OR = 6.4; CI = 2.5-16.4), in those that had undergone recent hospitalization (OR = 2.2; CI = 1.0-4.4), and it reached OR = 4.0 (CI = 1.7-9.9) in those with three or more antibiotic treatments in the previous year; about 10% of the variability in MRSA carriage could be attributed to differences between wards. Pulsed field gel electrophoresis analysis permitted the definition of six clusters; two of these comprised 78.6% of the studied isolates and were quite similar, with one being more strongly represented among subjects hospitalized in the previous 12 months. All of the MRSA strains were resistant to ciprofloxacine; nevertheless, the majority were susceptible to most other non-betalactam antibiotics. CONCLUSION: The study suggests that nursing homes are a significant reservoir for MRSA. Statistical and PFGE analyses indicate a scenario where MRSA seems to be endemic and individual risk factors, namely recent hospitalizations and repeated antibiotic treatments, play a major role in the selection of drug-resistant organisms. Infection control measures should be coordinated among different health care settings, and the appropriate use of antibiotics has emerged as an important issue for improving the quality of care.

Decrease of replicative capacity of HIV isolates after genotypic guided change of therapy.

A longitudinal study of the replication capacity of HIV strains isolated from 18 patients failing highly active antiretroviral therapy (HAART) was undertaken at the time of genotypic guided change of therapy and after 12 months. Patients were divided in two groups according to the response to therapy: immune responders (12 patients with immune recovery defined as having more than 100 CD4 cells compared to baseline value), and failing patients (six patients without immune recovery). At enrollment no significant difference in terms of CD4 cell count and HIV plasma viremia was detected between the two groups. One year after change of therapy, all patients experienced a decrease in the replication capacity of HIV strains. The HIV replication capacity of the failing and of immune-responder patients decreased from 60% (range 14-96%) to 26.4% (range 0.4-74.5) and from 46.8% (range 15-98%) to 3.6% (range 0.1-26.8%), respectively. At month 12, the difference of HIV replication capacity between the two groups reached a statistical significance (P<0.03). After the change of therapy, an increase in the number of drug resistance mutations in the protease gene was detected in both groups with a higher prevalence of M36I mutation in immune responders. The HIV strains of patients failing HAART showed a progressive impaired replication capacity. The degree of the impairment in viral replication correlated with the viro-immunological discordant response to HAART and with the acquisition of new drug resistant mutations in the protease gene. In patients failing HAART, the impaired replication capacity of HIV strains could justify the persistence of an immune recovery.

Failure of stavudine-lamivudine combination therapy in antiretroviral-naive patients with AZT-like HIV-1 resistance mutations.

To analyze the clinical relevance of AZT resistance mutations in AZT-naive patients, 56 HIV-1 seropositive patients treated for 18 months with stavudine/lamivudine (27 patients) or AZT/lamivudine (29 patients) were studied. AZT-like resistance mutations were found in 13 out of 29 (44%) patients treated with AZT/lamivudine and in 11 out of 27 (40%) patients treated with stavudine/lamivudine. No stavudine or multi-drug resistance mutations were detected. After 26 months of treatment more than 60% of patients showed a virological failure. Among 10 patients failing treatment with stavudine/lamivudine, 9 had AZT-like resistance mutations. The phenotypic test, performed on HIV-1 strains isolated from six of these nine patients, showed a resistance to AZT in five isolates and to stavudine in two isolates. The genotypic pattern of the latter two isolates showed the combined mutations M184V plus R211K and L214F. AZT-like resistance mutations in AZT-naive patients seem to correlate with a virological failure during long-term stavudine therapy.

Neutralizing antibodies against autologous human immunodeficiency virus Type 1 isolates in patients with increasing CD4 cell counts despite incomplete virus suppression during antiretroviral treatment.

Antiretroviral-treated human immunodeficiency virus (HIV) type 1-seropositive individuals can remain clinically stable for a long period of time with an increasing CD4 cell count irrespective of incomplete viral suppression. We evaluated the role of neutralizing antibody (NtAb) activity in the etiopathogenesis of this viro-immunological disconnection (defined as an increasing CD4(+)-cell count despite a persistent, detectable viral load during antiretroviral therapy) in 33 patients failing therapy with two analogue nucleoside reverse transcriptase inhibitors. An HIV NtAb titer of >/=1:25 was detected in specimens from 16 out of 33 (48%) patients. A significant correlation was found between NtAb titers and CD4(+)-cell counts (P = 0.001; r = 0.546) but not with HIV RNA levels in plasma. Five patients with a viro-immunological disconnection had an NtAb titer of >1:125, statistically higher than the NtAb titers for the remaining 28 patients with both virologic and immunologic failure (P < 0.0001). The HIV-specific humoral immune response could play a role during antiretroviral treatment to improve immunological function despite an incomplete suppression of viral load.

Cellular proviral HIV-DNA decline and viral isolation in naïve subjects with <5000 copies/ml of HIV-RNA and >500 x 10(6)/l CD4 cells treated with highly active antiretroviral therapy.

OBJECTIVE: To evaluate the decay rate of cellular proviral HIV-DNA and viral replication in patients receiving highly active antiretroviral therapy (HAART) in the very early phase of infection. METHODS: Thirty-four patients treated with HAART and retrospectively selected for progressive decline of plasma viraemia up to undetectable levels (< 20 copies/ml), were stratified according to CD4+ cell count and plasma viraemia at base line: > 500 x 10(6) cells/l with < 5000 copies/ml (group 1) or with > 5000 copies/ml (group 2), > 5000 copies/ml with 300-500 x 10(6) cells/l (group 3) or with < 300 x 10(6) cells/l (group 4). Plasma HIV-RNA and proviral HIV-DNA were analysed at baseline and after 1, 2, 3, 6, 9 and 12 months of treatment. RESULTS: After 1 year of treatment, a significant decrease of proviral DNA titre was observed in all patients and a decrease > 1 log was achieved in 24 of 29 subjects of the first three groups. The more pronounced decay of HIV-DNA (half-life 28 weeks) up to < 50 HIV-DNA copies/10(6) CD4+ cells was detected in patients of group 1. At the year's endpoint, five patients (four in group 1 and one in group 2) had < 20 HIV-DNA copies. However, HIV strains sensitive to antiretroviral drugs were isolated from peripheral lymphocytes of 16 out of 34 patients. CONCLUSION: In patients with undetectable plasma viraemia after 1 year of HAART, the highest reduction of proviral DNA up to < 50 copies/10(6) CD4+ cells was obtained only in subjects in the early asymptomatic phase of infection. Nevertheless, a replication-competent virus can be detected in all phases of antiretroviral therapy.

Saquinavir delays the emergence of zidovudine resistance in HIV-1 seropositive patients treated with combination therapy.

During a randomized double-blind study to assess the antiviral activity of saquinavir (SQV) alone or in combination with zidovudine (ZDV), the emergence of phenotypic resistance was evaluated in 44 patients treated with SQV (13 subjects), ZDV (14 subjects), and SQV plus ZDV (17 subjects). A significant (P< 0.05) lower CD4+ cell count and higher HIV RNA copy number at entry were found in six patients who developed resistant viral strain (3 to ZDV and 3 to SQV) during the first 4 months of treatment. After 1 year, drug-resistant strains (12 to ZDV and 14 to SQV) were isolated in 26 out of 37 patients. A significant higher number of patients treated with ZDV alone (10/13) harbored ZDV-resistant strains compared to patients treated by combination therapy (2/13); whereas more than 50% of patients had SQV-resistant strains aside from treatment. Early SQV-resistant strains were isolated in a limited number of patients treated with SQV alone (3/13). The rates of emergence of resistant strains during ZDV or SQV monotherapies are comparable. Combination therapy may delay the emergence of phenotypic resistance to either drugs in the short term and to ZDV, but not to SQV, at least after 1 year.

Five human immunodeficiency virus type 1 phenotypic variants with different MT-2 cell tropisms correlate with prognostic markers of disease.

OBJECTIVES: We correlated virologic and immunologic parameters of disease progression with cytopathogenicity of HIV isolates. STUDY DESIGN/METHODS: Human immunodeficiency virus type 1 (HIV-1) isolates from 207 patients with CD4+ cell counts < 500/mm3 were examined for biologic phenotype in MT-2 cells. We used a cross-sectional study design. RESULTS: Three subtypes of syncytium-inducing (SI) strains with different times of appearance of syncytia formation in cell culture and two subtypes of non-syncytium-inducing (NSI) isolates, with (NSI/MT2+) or without (NSI/MT2-) replicative capacity in MT-2 cells, were identified. Early SI strains were associated with the lowest CD4+ cell counts and the highest levels of viral load, and NSI/MT2- isolates correlated with the highest CD4+ cell counts and the lowest viral loads. Patients with late SI and NSI/MT2+ strains showed minimal differences in immunologic and virologic markers. CONCLUSIONS: Five HIV phenotypic variants that correlate significantly (P < 0.001) with markers of disease progression were identified.

Human immunodeficiency virus infection of human bone marrow stromal myoid cells.

In order to investigate the potential susceptibility of bone marrow stromal myoid cells to human immunodeficiency virus (HIV), a myoid cell population devoid of all other cellular components of marrow environment was isolated from 3 normal adult bone marrow samples. The bone marrow myoid cells were infected with 3 different strains of HIV-1, 2 strains isolated from patients with acquired immune deficiency syndrome (AIDS) and HTLV-IIIB strain. To demonstrate successful infection and HIV production, culture supernatants, harvested weekly until 2 months post-infection, were tested for the presence of p24 antigen and infectious virus. Myoid cell monolayers obtained from the 3 different bone marrow samples were shown to be susceptible to infection. In particular, infection led to the presence of p24 antigen and of infectious virus in culture supernatants up to day 49 post-infection. After day 49, it was not possible to demonstrate the presence of infectious virus in culture supernatants and HIV-DNA polymerase chain reaction (PCR) failed to show viral genome in any of the cultures assayed. Our results demonstrate the susceptibility of myoid stromal cells to HIV infection and may provide an in vitro model for studying the effects of HIV infection in disregulation of the haemopoietic function of bone marrow environment.

High rate of HIV isolation from plasma of asymptomatic patients through polyethylene glycol (PEG) treatment.

The usefulness of traditional methods of HIV plasma titration has been limited by poor detection capacity in the asymptomatic phase of HIV disease. We analyzed plasma samples from asymptomatic seropositive or early symptomatic patients, comparing the classic plasma culture method with the following techniques: phorbol 12-myristate 13-acetate treatment of target cells, centrifugal inoculation of the virus, heat treatment of cultures, addition of monocyte/macrophages to cultures, and polyethylene glycol (PEG) treatment of the plasma. Only PEG treatment significantly increased the percentage of HIV isolation. The increase of HIV isolation after PEG treatment is more evident in patients with higher CD4+ cell counts and those without detectable levels of p24 antigen. In the p24-negative samples, HIV was isolated in 17 of 24 (71%) with PEG treatment versus nine of 24 (37%) with the classic method (p < 0.01). A number of discordant samples were found using the classic and PEG methods. Combining the positive results obtained with either technique, we obtained an overall HIV detection rate of 76%. The increased sensitivity of the combination of PEG and classic methods may allow a wider use of plasma viremia as part of the virological evaluation of anti-HIV drug efficacy in asymptomatic patients.