Myeloablative conditioning was associated with a lower risk of AIC compared with reduced intensity conditioning (RIC) in fludarabine and/or alemtuzumab  and ATG containing regimens . These findings provide clinically useful information regarding the incidence of a rare and potentially life-threatening complication of allogeneic HSCT for aAA, and further support for BM as the preferred stem cell source for transplant of patients with aAA.This retrospective study explored the incidence of autoimmune cytopenia (AIC) in 530 paediatric and adult patients with acquired aplastic anaemia (aAA) who underwent first allogeneic HSCT between 2002 and 2012. AIC was a rare complication with a cumulative incidence of AIC at 1, 3, 5 and 10 years post HSCT of 2.5% (1.2–3.9 95% CI), 4.4% (2.6–6.2 95% CI), 4.6% (2.8–6.5 95% CI) and 5.1% (3.1–7.2 95% CI). Overall survival at 5 years after diagnosis of AIC was 85.9% (71–100 95% CI). Twenty-five patients were diagnosed with AIC at a median of 10.6 (2.6–91.5) months post HSCT. Eight (32%) patients were diagnosed with immune thrombocytopenia (ITP), seven (28%) with autoimmune haemolytic anaemia (AIHA), seven (24%) with Evans syndrome and four (16%) with autoimmune neutropenia (AIN). Treatment strategies were heterogeneous. Complete responses were seen in 12 of 25 patients, with death in three patients. In multivariable Cox analysis of a subgroup of 475 patients, peripheral blood stem cell (PBSC) transplant was associated with higher risk of AIC compared with bone marrow (BM) when conditioning regimens contained fludarabine and/or alemtuzumab (2.81 [1.06–7.49 95% CI];  Graft versus host disease (GvHD) describes alloreactivity between a transplanted donor immune system and host major or minor histocompatibility antigen, and is the principal immune mediated complication of allogeneic haematopoietic stem cell transplant (HSCT). The phenomenon of post-HSCT autoimmune disease (AID) is also recognized, and is the pathological autoreactivity towards non-histocompatibility antigen common to donor and recipient. Reported transmission of autoantibodies and AIDs from donor to recipient, and a case of immune thrombocytopenia (ITP) in the context of syngeneic HSCT suggest that this is a pathological entity distinct from GvHD , 2. WhetThis study was conducted in accordance with the Declaration of Helsinki, and approved by the EBMT Autoimmune Disease Working Party and Severe Aplastic Anaemia Working Party. All EBMT centres performing allogeneic HSCT for aAA were invited to participate in the study. Centres agreeing to participate provided data on all consecutive paediatric and adult patients with aAA treated between January 2002 and December 2012 with first allogeneic HSCT. Centres confirmed whether AIC was diagnosed post-HSCT. Patients diagnosed with AIC at any time point prior to HSCT were excluded. Data for all patients were extracted from the EBMT registry, and centres identifying cases of AIC provided additional data on diagnostic criteria and investigations undertaken, therapies administered and patient outcome.The primary study objective was to estimate the incidence of AIC diagnosed after allogeneic HSCT for aAA. Secondary objectives were to identify patient, donor and HSCT-related risk factors for development of AIC; to scope diagnostic criteria for AIC used across EBMT centres; to explore treatment strategies and response; and to estimate overall survival (OS) following onset of AIC.9/L, a new or worsening anaemia with a fall in haemoglobin of ≥20 g/L and new or worsening neutropenia <1.5 × 109/L respectively. Centres were asked to indicate further laboratory features that had established an AIC diagnosis. Lines of treatment are presented as reported by participating centres. Complete response was defined as a normalisation of haemoglobin, platelet or neutrophil count and normalisation of biochemical markers of haemolysis for AIHA and Evans syndrome. Partial response was defined as an improvement but not normalisation, and no response as either no improvement or a decline in these parameters.ITP, autoimmune haemolytic anaemia (AIHA) and autoimmune neutropenia (AIN) were defined as a new or worsening thrombocytopenia with a platelet count of <100 × 10p-values were two-sided and p < 0.05 was considered significant. Statistical analyses were performed using SPSS version 22  and R version 3.0.3.Pre-transplant patient characteristics were expressed as the median and range for continuous variables and frequencies and proportions for categorical variables. The primary endpoint was the incidence of post-transplant AIC, evaluated at 1, 3, 5 and 10 years after transplant. OS for the study cohort was evaluated at 1, 3, 5 and 10 years after transplant. OS after AIC diagnosis was analysed separately and evaluated at 5 years after AIC diagnosis. Median follow-up was determined using the reverse Kaplan–Meier method. OS was estimated using the Kaplan–Meier product limit estimation method, and differences in subgroups were assessed by the log-rank test. Cumulative incidences of AIC and death without AIC were analysed together in a competing risks framework. Subgroup differences were assessed using Gray’s test. The effect of specific conditioning regimens on the incidence of AIC and mortality without AIC were investigated using multivariable Cox models, providing cause specific hazards. The association between chronic GvHD and AIC was explored using a time dependent Cox model. A landmark analysis was used to compare incidence of AIC between those patients with and without acute GvHD. All reported estimates include 95% confidence intervals. All A total of 530 patients  from 41 participating centres were eligible for inclusion in the study. Median age at HSCT was 21.4 years (1.7–69.8). Median follow-up time was 6.4 years (interquartile range 4.2–9.0), and 1, 5 and 10-year OS for the cohort was 85.3% (82.3–88.4 95% CI), 80.8% (77.4–84.2 95% CI) and 78.8% (75.0–82.6 95% CI).n = 8) ITP, 28.0% (n = 7) AIHA, 24.0% (n = 6) Evans syndrome  and 16.0% (n = 4) AIN , 4.4% (2.6–6.2 95% CI), 4.6% (2.8–6.5 95% CI) and 5.1% (3.1–7.2 95% CI), respectively. OS at 5 years after diagnosis of AIC was 85.9% (71–100 95% CI), with all deaths occurring within the first 12 months.9/L. BME was performed in 6/8 patients, with four displaying normo- or hyper-regenerative megakaryopoiesis, and two other morphological or histological findings. Five centres indicated that differential diagnoses  had been excluded, two centres reported that this was unknown and one that all differential diagnoses had not been excluded. All AIHA patients had a new or worsening anaemia with a fall in haemoglobin of ≥20 g/L. Direct antiglobulin test (DAT) was performed in 6/7 cases with one IgG positive, and five IgG and C3d positive  samples was not possible. In addition to reporting diagnostic criteria, centres were asked to report whether all differential diagnoses had been excluded. It was decided a priori to include all reported cases of AIC in study analysis, and describe the diagnostic features employed by participating centres in order to present real-world practice during the study period. For ITP, all patients had a new or worsening thrombocytopenia with a platelet count of <100 × 10Of 25 patients diagnosed with AIC, 21 (84%) were treated, while 2 (8%) ITP (Cases 1 and 2) and 2 (8%) AIN patients (Cases 22 and 23) received no AIC specific therapy  relapsed and required further treatment. AIC status at last treatment was 4/25 untreated, 12/25 CR, 6/25 in PR and 3/25 NR. Six out of twenty one treated patients required on-going maintenance therapy (Table p = 0.3). The incidence of limited and extensive cGvHD was 20% (16–24 95% CI) at both 60 and 120 months. There was no association between cGvHD and development of AIC . We did not evaluate the temporal relationship between withdrawal of immunosuppression and onset of AIC. Ten-year cumulative incidence of AIC was higher amongst patients treated with non-myeloablative conditioning (MAC) compared with MAC , with conditioning regimens containing fludarabine compared with regimens not containing fludarabine  and with alemtuzumab containing regimens compared with non-Alemtuzumab containing regimens . Conditioning with anti-thymocyte globulin (ATG) was associated with a lower incidence of AIC than conditioning without ATG . Incidence of AIC was also significantly higher in patients receiving peripheral blood stem cell (PBSC) (9.7% [4.7–14.8 95% CI]) compared with umbilical cord blood (UCB) (5.0% [0.0–14.6 95% CI]) and bone marrow (BM) (3.3% [1.2–5.6 95% CI]) (p = 0.01). Incidence of AIC was not higher in regimens containing alemtuzumab and fludarabine compared with alemtuzumab alone , nor in regimens containing ATG and fludarabine compared with ATG alone .In univariable analysis, cumulative incidence of AIC at 10 years post HSCT did not vary with patient sex, patient age group, interval from aAA diagnosis to HSCT, patient cytomegalovirus (CMV) status, donor type, patient-donor CMV match, patient-donor sex match or conditioning with TBI (Table p = 0.723). However, after adjustment for alemtuzumab and/or fludarabine, transplant with PBSC remained independently associated with a higher risk of AIC , and MAC with lower risk . Likewise, in the multivariable ATG model, use of this serotherapy in conditioning was not associated with higher risk of AIC  but PBSC  and MAC  remained independently associated with higher and lower risk, respectively. There was no increase in the competing risk, death without AIC, associated with any of these variables. Owing to small number of AIC cases it was not possible to adjust for additional characteristics and so these findings are not generalizable beyond the study population.A subgroup of 475 patients who received either BM or PBSC grafts and had complete information on conditioning regimen were included in a multivariable analysis. Two models explored the independent association of conditioning intensity, stem cell source, use of alemtuzumab and/or fludarabine or use of ATG with risk of AIC Table . In the Ten-year cumulative incidence of AIC after allogeneic HSCT for aAA was 5.1% 3.1–7.2 95% CI). ITP was the most frequent AIC diagnosis, followed by AIHA, Evans and AIN. BME was not performed in two patients diagnosed with ITP. In a further two patients BME did not demonstrate normo- or hyper-regenerative megakaryopoiesis. It is possible that these patients were misdiagnosed biasing our results. AIC complicated both the early and late post-HSCT period, with time of diagnosis ranging from 2.6 months to 7.6 years. Incidence of AIC in our cohort is similar to that reported in previous studies of HSCT populations. In paediatric patients transplanted with a range of allogeneic graft types for malignant and non-malignant conditions, a 2.4–2.5% 10-year incidence of AIC, and a 2.4% incidence of AIHA are reported . Among a.2 95% CICases of AIC in our study were diagnosed in accordance with recognized criteria –14. In tMost ITP patients were treated with corticosteroid or IVIg, while rituximab was infrequently used. Fifty percent of ITP patients achieved either CR or PR with each line of therapy. In the general population a response to first-line steroids of up to 80% has been reported , 18, andAlthough randomized data are lacking, corticosteroids are an established first-line therapy for AIHA with retrospective studies reporting responses of ~75% . Again, Primary AIN is typically a disease of infancy, while secondary AIN occurs mostly in adults in the context of systemic AID . NeutropThe pathogenesis of AID remains poorly understood but relates to breakdown in mechanisms of self-tolerance. First, there may be a genetic role, with documented familial aggregation of autoimmune conditions , the assIn allogeneic HSCT for aAA, BM conveys a lower risk of GvHD and a survival advantage compared with PBSC, and is the preferred stem cell source in all age groups and in recipients of sibling and unrelated donor transplants –56. NoneThis is the first study to explore AIC following allogeneic HSCT in a large cohort for a single disease indication, and provides clinically useful information on the incidence of this rare and potentially life-threatening complication in paediatric and adult patients. The retrospective study design may impact on our estimate of AIC incidence: cases may not have been reported and we were unable to confirm AIC diagnoses through central review of pathology. For eight patients with AIC, centres reported that it was unknown whether all differential diagnoses had been excluded, and for 3 that all differentials had not been excluded. These cases were included in analysis to reflect real-world practice and highlight the challenging nature of establishing these diagnoses, but it remains possible that these patients were misdiagnosed biasing our results. As there were relatively few cases of AIC, we were able to develop a multivariable model with a small number of variables that limits generalisability of our findings. We did not explore the role of pre-transplantation therapy on risk of developing AIC.In conclusion, the incidence of AIC post allogeneic HSCT for aAA is similar to that reported among recipients of HSCT for other primary diseases. Autoimmunity should be considered in the differential diagnosis of cytopenias occurring in both the early and late post HSCT period. Our study highlights heterogeneity in the approach to investigation and management of AIC between EBMT centres. As such standardization of diagnostic and therapeutic approaches, along with data registration (via the EBMT and other databases), is warranted to inform harmonized recommendations for investigation and management of suspected AIC. Response rates in this cohort are similar to previous data and OS in the AIC group was not shorter than in the overall cohort. In multivariable analysis we identified an increased risk of AIC following PBSC and RIC HSCT in this cohort, which may contribute further to the evidence that BM is the preferred stem cell source for transplant of patients with aAA.


Vibrio parahaemolyticus (A3 and D4), strains known as having potentially harmful health consequences for aquatic crustaceans and consumers. Herein, we report that a natural antimicrobial mixture (A3009) was capable of significantly reducing the virulence of V. parahaemolyticus strains A3 and D4 in an in vitro infection model, using the fish cell line CHSE-214, an effect which correlates with the bacterial downregulation of hcp1 and hcp2 gene expression and with the ability of the antimicrobial to efficiently retain low cytotoxic levels (p < 0.001). We show for the first time that a natural antimicrobial is able to significantly reduce the mortality of shrimps in a challenge experiment and is able to significantly attenuate H2O2 release during infection (p < 0.001), indicating that it could harbor positive intestinal redox balance effects.Reducing acute mortality in aquatic crustaceans using natural alternatives to antibiotics has become a necessity, firstly for its positive impact on the aquaculture industry and, secondly, because the extensive use of antibiotics may lead to increased levels of drug resistance in pathogenic microorganisms. This study aimed to investigate the effect of a mixture of natural antimicrobials on the in vitro and in vivo virulence abilities of Type VI secretion system (T6SS)-positive  V. parahaemolyticus is a Gram-negative bacterium, halophilic in nature with a typically rigid curve or straight non-spore forming rod shape [Vibrio genus are commonly recognised as inhabitants of fresh and marine waters, with over 100 species being described so far [V. parahaemolyticus has not been shown to do so [V. parahaemolyticus is a toxin producing bacterium, in which the toxins are controlled by the genes pirA and pirB and together are responsible for acute hepatopancreatic necrosis disease (AHPND) [V. parahaemolyticus [od shape . The bacd so far . Bacterito do so . V. para (AHPND) . Individ (AHPND) . This sy (AHPND) . AHPND i (AHPND)  and has  (AHPND) . Studiesolyticus .Vibrio cholerae [hcp), an important virulence factor in many bacteria, including Pseudomonas aeruginosa [Campylobacter spp. highlighted that the T6SS system is involved via the downregulation of hcp gene expression in the reduction of the bacterium’s ability to adhere and infect in vitro [Campylobacter spp. In V. parahaemolyticus, the hcp haemolysin signature gene has two proteins, hcp1 and hcp2, which interestingly are expressed under different growth conditions mainly related to salt concentration (1.5–3% NaCl) [hcp1 and hcp2 and their dependency on the salt concentration seems to be related to the ability of the pathogen to compete for a niche, especially in a marine environment, and to enhance their virulence ability [The Type VI secretion systems (T6SS) are associated with severe forms of gastrointestinal diseases and have been described mostly in Gram-negative bacteria . Bactericholerae  and it wruginosa . Infectiin vitro . The sam3% NaCl) . The dif ability .V. parahaemolyticus detection strategies are continually being developed in an attempt to reduce the risk of infection via seafood consumption [V. parahaemolyticus in contaminated seafood and prevent potential outbreaks [Vibrio species [sumption ,16. Variutbreaks . Howeverutbreaks . Antibioutbreaks . Despiteutbreaks . The lar species . V. parahaemolyticus strains A3 and D4 in vitro and in vivo and identify the virulence factors involved. We also aimed to investigate their potential to attenuate V. parahaemolyticus virulence by using sub-lethal concentrations of the antimicrobial mixture as there is a constant concern that bacteria could also gain resistance to natural antimicrobials [This study aimed to investigate how a mixture of natural antimicrobials could regulate the growth of crobials .Vibrio parahaemolyticus A3 (origin Vietnam) and D4 (origin Mexico) strains were kindly donated by Kim Orth from the Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Both strains were routinely cultured in marine Luria–Bertani (MLB) broth (Luria–Bertani broth containing 1.5% or 3.5% NaCl). The CHSE-214 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC). The CHSE-214 cells (ECACC No. 91041114) are a fish cell line derived from Oncorhync hustshawytscha embryos. They were cultured in minimum essential medium (MEM)  supplemented with 10% foetal bovine serum , 2 nM l-glutamine , and an antibiotic/antimycotic solution . A mixture of antimicrobials containing lactic acid, E330 citric acid and citrus extract was used. The composition (per kg) of the feed material was sodium chloride (1 g) and maltodextrin. The additives (per kg) were preservatives, including lactic acid and E330 citric acid, and the flavouring was citrus extract. The analytical constituents (%) were crude ash (0.2%), crude protein (0.84%), crude oils and fats (10.83%), crude fibre (0.03%), phosphorus (0.01%), sodium (1.12%), calcium (1.34%), lysine (0%) and methionine (0%). The antimicrobial product  was obtained from Envirotech Innovative Products Ltd. The Vibrio parahaemolyticus, strains A3 and D4. The strains were grown overnight at 37 °C in nutrient broth  containing 1.5% or 3% NaCl. The resulting stationary phase cultures were diluted using a nutrient broth containing 1.5% or 3% NaCl, to give a suspension containing approximately 6 log CFU/mL A series of Auranta 3009 solutions were prepared in a nutrient broth containing 1.5% or 3% NaCl, to give a range of concentrations from 4% down to 0.015625%. One ml of each Auranta 3009 solution was transferred into separate sterile plastic bijou bottles and 1 mL of either strain of V. parahaemolyticus was added to each bottle. A positive control containing 1 mL of nutrient broth and 1 mL of the 6 log CFU/mL suspension was prepared. The negative control was 2 mL of uninoculated nutrient broth plus salt. The mixtures were incubated aerobically at 37 °C for 24 h. This procedure was repeated on three separate occasions. The MIC value was determined as the lowest concentration of Auranta 3009 that showed no bacterial growth. After 24 h, the bijou bottles were observed for bacterial growth. If the broth was clear, indicating no bacterial growth, then 100 µL was spread-plated onto nutrient agar containing 1.5% or 3% NaCl using a sterile glass spreader. This was repeated for all concentrations of Auranta 3009 that appeared clear in the bijou container. All plates were incubated aerobically at 37 °C for 24 h. This procedure was repeated on three separate occasions for both V. parahaemolyticus strains A3 and D4. The MBC value was determined as the lowest concentration of Auranta 3009 that showed no bacterial growth on the plates.The minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MBC) were determined for two strains of acute hepatopancreatic necrosis disease (AHPND)-causing V. parahaemolyticus; A3 and D4. Broth cultures were prepared in a nutrient broth containing 1.5% or 3% NaCl and incubated overnight at 37 °C. These were diluted using nutrient broth containing 1.5% or 3% NaCl, to give a final concentration of approximately 6 log CFU/mL. The MIC value for both V. parahaemolyticus A3 and V. parahaemolyticus D4 was previously determined to be 0.0625% of Auranta 3009. An Auranta 3009 solution was prepared using a nutrient broth containing 1.5% or 3% NaCl to give a final concentration of 0.0625% when the suspension of bacteria was added. Two-fold dilutions of this solution were prepared in a 96-well plate giving a range of Auranta 3009 concentrations from the MIC (0.0625%) down to 1/8 of the MIC concentration (0.0078125%). The final volume of Auranta 3009 solution in each well was 90 µL. The bacterial suspension (10 µL) was added to each well and thoroughly mixed. Nutrient broth containing 1.5% or 3% NaCl with no Auranta 3009 added was inoculated with the bacterial suspension as a positive control. The negative control was uninoculated nutrient broth containing 1.5% or 3% NaCl. The 96-well plate was sealed with plastic film and the optical density was measured at 600 nm at intervals of 1 h over a 24 h period at 37 °C using a FLUOstar Omega automatic plate reader . This procedure was repeated on three separate occasions for both Vibrio parahaemolyticus A3 and D4.Growth curves were established for both strains of V. parahaemolyticus strains A3 and D4 were selected to be tested for their pathogenicity by a challenge test using healthy Penaeus vannamei post larvae, following a procedure previously described [9/mL bacteria for 10 min. The antimicrobial mixture was applied at the time of infection in concentrations of 0.0625%, 0.031%, 0.015% and 0.007% in 500 mL flasks. Survival was determined by counting the larvae at 30 h after infection. A positive and a negative control  was also included in the challenge at 0% of the antimicrobial mixture. The experiment was performed in triplicate. escribed . Twenty-V. parahaemolyticus strains A3 and D4 was prepared as described above. The CHSE-214 cells were grown in a minimum essential medium  -Gibco®, Waltham, MA, USA) until they reached confluence and were supplemented with 10% fetal bovine serum and 2 mL glutamine. The cells were routinely grown in 75 cm2 tissue culture flasks , in a humidified incubator at 37 °C with 5% CO2. The H2O2 production from infected and un-infected CHSE-214 in response to treatment with Auranta 3009 at was measured using the PeroxiDetect™ Kit , following manufacturer guidelines and as described in [A culture mixture of ribed in . V. parahaeomolyticus strains A3 and D4 the isolates were grown to the OD600 of 0.5. The CHSE-214 were cultured as described above. Prior to the infection of the cells, the cell monolayers were incubated for 3 h with tissue culture media containing 0.031% Auranta 3009 followed by washing three times with the tissue culture media, and then infected for 3 h with 104 CFU/mL V. parahaemolyticus strains A3 and D4. After infection, the infection media was removed and the infected monolayers were washed three times with the tissue culture media. The infected cells were then incubated with tissue culture media containing gentamicin (100 µg/mL) in order to expose the internalised bacteria or to total lysis without gentamicin inclusion, but, instead, 0.1% Triton X was included to reveal the total bacterial adhesion. In order to determine the role of hcp1 in marine conditions (3% NaCl), in V. parahaeomolyticus infection, we have co-cultured the microorganism in nutrient broth containing 1.5% or 3% NaCl in the presence of 0.031% Auranta 3009 for 3 h. The co-cultured microorganisms were then used in the infection assay at an MOI of 100. Similarly, the experiment was also performed to investigate the effect of hcp2 expression, but this time with bacteria grown in low salt concentrations (1.5% NaCl). The bacteria grown in the above conditions was also used to determine the hcp1 and hcp2 gene expression in the above conditions. The primers used to detect hcp1 were: cacgtgacggctcggtgg and ctcttctttcgcgtcttggtcg and for hcp2 cgagtatccactcgaaactttc and ttctgctccctcagtactttctg [® Green PCR Kit  on a LightCycler® 96 . A total of 5 μL of SYBR Green master mixture was used in each reaction, along with 0.5 μL of 10 μM primer mixture, 3 μL of molecular grade water, and 1 μL of DNA sample. For hcp 1 and 2 , a total of 5 μL of SYBR Green master mixture was used in each reaction along with 0.8 μL of 20 μM primer mixture, 7.4 μL of molecular grade water, and 1 μL of DNA sample. The relative quantity of the mRNA was calculated using the ΔCt method. The rARN 16S gene was used as an endogenous control since it was transcribed in equal rates in both the treated and untreated cells.To confirm the efficacy of Auranta 3009 in reducing the adhesion and invasion abilities of V. parahaemolyticus strains A3 and D4. The direct impact of Auranta 3009 was investigated by pre-treatment of CHSE-214 cells with 0.031% Auranta 3009 for 3 h. After 3 h, the media was removed, the cells were washed 3× with fresh MEM, followed by infection with V. parahaemolyticus strains A3 and D4 at an MOI of 100. The CHSE-214 cells were also infected with pre-treated bacteria. Uninfected cells exposed to 0.031% of Auranta 3009 were used as controls. Lactate dehydrogenase release (LDH) was measured with a cytotoxicity detection kit  following the manufacturer’s instructions. The results are expressed as cytotoxicity calculated as a percentage of the total lysis of cells lysed in Triton X 100.The CHSE-214 cells were infected as described above over a 4 h time course with V. parahaemolyticus strains A3 and D4 were co-cultured in nutrient broth containing 1.5% NaCl or 3% NaCl in the presence of 0.031% Auranta 3009 for 3 h. The co-cultured organisms where then placed (~1 × 106 CFU) at the centre of the agar plate. The diameter of the motility area was measured. Non-co-cultured strains were used as a control. The experiments were performed in triplicate and on three separate occasions. Motility was assessed as previously described . BrieflyV. parahaemolyticus strains A3 and D4 in the presence of 1.5% and 3% NaCl. The experiment was designed to identify the concentration at which the antimicrobial mixture has no lethal effect on the V. parahaemolyticus strains A3 and D4. This concentration was required to allow us to investigate the possible alteration of bacterial virulence factors while the pathogens are still alive. The MIC and MBC values at levels of 1.5% and 3% NaCl against V. parahaemolyticus A3 and V. parahaemolyticus D4 are shown in First, we aimed to gain insight into the growth characteristics of V. parahaemolyticus A3 and D4 motility, a major virulence factor, at the sub-inhibitory concentration calculated at half of the MIC (0.031%) in the presence of 1.5% and 3% NaCl (2 concentration (∗∗∗ p < 0.001). These results suggested that the antimicrobial mixture reduces the bacterial motility and potentially has an impact on virulence.We next investigated the potential impact of the antimicrobial mixture on the  3% NaCl . The motV. parahaemolyticus A3 and D4 hcp gene expression is shown in p < 0.001) in hcp1 gene expression levels in the presence of 3% NaCl in V. parahaemolyticus A3, with no effect on hcp2 at this salt concentration. The downregulation effect on hcp2 was only observed in the presence of 1.5% NaCl, as indicated in p < 0.001). The results were similar for V. parahaemolyticus D4 in regard to the downregulation effect on hcp1 (∗∗ p < 0.01) and hcp2 (∗∗∗ p < 0.001) gene expression produced by Auranta 3009 at 1.5% and 3% NaCl . The impact on bacterial internalisation was as significant as for adhesion for both strains, as indicated in V. parahaemolyticus A3 and panel D for V. parahaemolyticus D4 (p < 0.001). When the antimicrobial mixture was only present in the tissue culture media during the adhesion/invasion assay, the reductions followed the same pattern (data not shown). These results indicate that the reduced in vitro infection abilities of V. parahaemolyticus A3 and D4 follow a similar pattern and in concordance with the reduction in bacterial motility.In order to correlate the negative effect on motility and a possible decrease in virulence, we performed in vitro infection assays using the CHSE-214 cell line as described in Materials and Methods. The pre-treatment of ticus A3 , panel Aticus A3 , panel CP. vannamei shrimps with V. parahaemolyticus strains A3 and D4. Our results show that at the MIC/MBC concentration of the antimicrobial (0.0625%) the lowest shrimp mortality was recorded  by the presence of Auranta 3009 (2O2 levels similar to the control. We next investigated the impact of nta 3009 . No effeV. parahaemolyticus A3 (p < 0.001). Following exposure to the 0.031% antimicrobial mixture, the infected CHSE-214 cells showed a clear reduction in cytotoxicity during infection with ticus A3 , panel Aticus A3 , panel BStaphylococcus aureus are inhibited by venom peptides from being promising candidates for the treatment of related infections caused by multi-drug resistant bacteria [H. pylori genes involved in colonisation [Vibrio strain infections in South American farmed shrimps [Natural antimicrobials are known to be effective in reducing bacterial pathogenicity via direct  or indirbacteria  Quorum-sbacteria . Anotherbacteria . They canisation . Antimic shrimps ; howeverV. parahaemolyticus is often found in estuaries and coastal waters and because it causes severe disease is also of significant importance as a human pathogen [V. parahaemolyticus), posing a threat to human and animal health and is of major concern to fish and shellfish farmers [V. parahaemolyticus and strains A3 and D4 [V. parahaemolyticus strains A3 and D4 [V. parahaemolyticus in such environments [pathogen . Phage apathogen ,33. Cons farmers ,35. Earl3 and D4  were pre3 and D4 . V. para3 and D4 . This deronments . V. parahaemolyticus to produce and release its toxin [Campylobacter jejuni, Campylobacter coli and Shiga Toxin Producing E. Coli (STEC) [Natural antimicrobials offer such an opportunity by being known to affect bacterial cell membranes . One sucts toxin . The anti (STEC) ,41. Thisi (STEC) ,42.C. jejuni RC039, where the downregulation of the hcp gene was associated with reduced virulence both in vitro and in vivo [According to our results, such mixtures can be used efficiently in both fresh and marine water environments, as we have observed similar results in low and high salt conditions. Similar antimicrobial mixtures have been previously used to prevent the bacterial infection of epithelial cells, both in vitro and in vivo , through in vivo . V. parahaemolyticus A3 and D4 appear to stimulate H2O2 release from human intestinal epithelial cells, possibly by altering the redox balance within the host’s lumen or by triggering the host NADPH oxidases. Microbiota-induced alterations to the lumen redox balance could have a modulatory effect upon pathogenicity, favoring the intestinal pathogen [2O2 release. However, the presence of hydrogen peroxide during the interaction between V. parahaemolyticus and the host epithelium does not seem to be critical, since it is able to significantly express catalase enzymes and induce a detoxification process [In addition, pathogen . Therefo process .V. parahaemolyticus A3 and D4 strains. The antimicrobials reduced the ability of V. parahaemolyticus A3 and D4 to infect the CHSE-214 fish cell line by inhibiting the expression of the T6SS hcp1 and hcp2 genes and through the reduction of bacterial motility in both low and high salt environments. These results corroborated with the antioxidant effect, low tissue cytotoxicity and the low mortality recorded in the in vivo challenge test, suggesting that this antimicrobial mixture (A3009) can be efficiently used to improve the survival of shrimp populations when subject to V. parahaemolyticus infections.The data presented herein describe the effect of a mixture of antimicrobials (A3009) on the virulence and pathogenicity of T6SS


Robot-assisted radical prostatectomy (RARP) is the most recent surgical technique for localized prostate cancer. The Da Vinci  system was first introduced in Brazil in 2008, with a fast growing number of surgeries performed each year. Our primary endpoint is to analyze possible predictors of functional outcomes, related to patient and tumor features. As secondary endpoint, describe functional outcomes  from RARP performed in the Sírio-Libanês Hospital (SLH), a private institution, in São Paulo, from April 2008 to December 2015.no pad on medical interview and sexual potency defined as the capability for vaginal penetration with or without fosphodiesterase type 5 inhibitors. Data from 104 consecutive patients operated by two surgeons from the SLH (MA and SA) between 2008 and 2015, with a minimum 12 months follow-up, were collected. Patient features , tumor features  and follow-up data  were the variables collected at 1, 3, 6 and 12 month and every 6 months thereafter. Continence was defined as the use of  Mean age was 60 years old and mean BMI was 28.45 kg/m2. BMI >30kg/m2 (p<0.001) and age (p=0.011) were significant predictors for worse sexual potency after surgery. After 1, 3, 6 and 12 months, 20.7%, 45.7%, 60.9% and 71.8% from patients were potent, respectively. The urinary continence was reached in 36.5%, 80.3%, 88.6% and 92.8% after 1, 3, 6 and 12 months, respectively. Until the end of the study, only one patient was incontinent and 20.7% were impotent. Age was a predictor of urinary and erectile function recovery in 12 months. BMI was significant factor for potency recovery. We obtained in a private hospital good functional results after 12 months of follow-up. Prostate cancer is the most common solid organ neoplasm in men and the second cause of cancer death in Brazil. Estimates of the National Cancer Institute (INCA) for the year 2016 predict 61,200 new cases. In 2013, 13,770 deaths by prostate cancer were confirmed , a private hospital in São Paulo, Brazil.All patients have been diagnosed with adenocarcinoma of the prostate by biopsy of the prostate guided by transrectal ultrasonography with at least 12 fragments. Only patients with clinically localized disease, life expectancy greater than 10 years and postoperative follow-up of at least 12 months, were included. Patients with previous history of radiotherapy or any neoadjuvant therapy were excluded.American Joint Committee on Cancer/Union for International Cancer Control), , sexual function prior to the procedure (by interview), PSA , prostate size – weighing of the piece performed by the pathologist (grams), date of surgery, comorbidities , Gleason of the surgical piece, pathological stage (2002 TNM of the ontrol),  sexual pSurgeries were performed according to the technique described by Patel . Most ofContinuous quantitative variables were described by measures of central tendency and dispersion, while categorical variables were described by means of absolute and relative frequencies (percentages).The time to reach potency and continence was assessed through Kaplan-Meier. The comparison of the curves was done by the log-rank test for categorical variables. Patients who did not reach potency / continence according to the pre-established criteria (see methods), until the last consultation, were considered impotent and incontinent.Cox regression models were constructed to identify independent predictors of urinary continence and sexual potency for continuous variables. Only the variables that reached p<0.25 in the analysis were included in the multivariate analysis.The results of the statistical tests were considered significant when p<0.05. All variables were entered into a database and analyzed using the R statistical program .2 (±4.2 kg/m2). The remaining descriptive  and the average BMI was of 28.45 kg/mcriptive  and catecriptive  characteData from 96 and 94 patients were analyzed for continence and potency, respectively. Two patients did not return to all the scheduled appointments and 6 patients had important data missing from their charts. Another 2 patients were excluded from potency analysis as they started androgen deprivation therapy (ADT) before 6 months of follow-up.None of the analyzed continuous variables  were shown to be related to continence recovery in the univariate analysis . However2 regained sexual potency.In the univariate analysis, BMI (p<0.001) and age (p=0.11) were statistically significant factors that influenced potency recovery. They also remained statistically significant in the multivariate analysis. Hypertension, diabetes, smoking, dyslipidemia and the presence of comorbidities also did not influence the recovery of sexual potency. The evaluation of age as a categorical variable wasn’t statistically significant.The frequency of continent patients immediately after removal of the bladder catheter and after 3, 6 and 12 months were of 36.5%; 80.3%; 88.6% and 92.8%, respectively. After the first year, 6 patients who were incontinent (6.2%) recovered urinary continence by the end of the evaluated period . Figure-Recovery of sexual potency occurred as early as the first month for 20.7% of patients. In the course of 3, 6 and 12 months, 45.7%; 60.9% and 71.8% recovered potency, respectively . The aveno pad) in this series. The average time for continence recovery was 2.66 months. Our continence results, at the end of the first year after surgery, are within the range observed in the systematic review and meta-analysis of Ficarra et al., which showed an average incidence of urinary incontinence at 12 months of 9% (8%-11%), despite considering continent patients using up to 1 pad per day  , age is 2) has also been reported as an adverse prognostic factor in radical prostatectomy. In a recent review study, BMI was responsible for longer surgical time, greater surgical bleeding and worse functional results (2), overweight (≥25 and <30 kg/m2) and obese (≥30 kg/m2) (2 and 91.4% of patients with BMI<30 kg/m2 continent (0 pad) after 6 months (p≤0.001) . This may be explained due to the small number of obese patients in the series.Obesity (BMI ≥ 30 kg/m results . Wiltz e0 kg/m2) . Patientp≤0.001) . In our After 1, 3, 6 and 12 months, there were 20.7%, 45.7%, 60.9% and 71.8% of patients who recovered sexual potency, respectively. After 12 months, another 7 patients regained sexual potency and inSexual Health Index for Men) questionnaire, a Canadian study with 722 cases reported recovery of sexual potency in 1, 3, 6 and 12 months of 19.5%; 31.4%; 37.2% and 52.4% (In 19 cases (20.7%) there was no recovery of sexual function. The average time to recover sexual potency was 7.72 months. Shikanov et al. reported similar results using the interview made by the surgeon. After 3, 6 and 12 months, results were 57%, 63% and 82%, respectively . Using Snd 52.4% .2 and <30kgm2) and normal patients (BMI<25kgm2), with 48.4%, 59.6% and 68.5% of potent patients after 12 months, and 55.9%, 78.9% and 80.3% after 24 months, respectively (p=0.02) (2 and erectile dysfunction 12 months after RARP (p<0.001) found in our study may be related to the prior committed sexual function and the presence of other comorbidities that were not evaluated.The relationship between BMI and sexual potency recovery after RARP is suggested in some studies, but it is still controversial , 26. It (p=0.02) . Wiltz e(p=0.02) . The assThe association between comorbidities that increase cardiovascular risk such as diabetes, hypertension, dyslipidemia, and smoking with erectile dysfunction is well recognized . Such diOur study presents a number of limitations inherent to a retrospective study. Data collected from medical and hospital records are not always complete and often underestimate complications and eventually overestimate the results. No specific urinary continence or sexual potency questionnaires were applied, which could provide more reliable data. Nevertheless, since we considered continent all patients who did not need any type of pad protection, we believe that the results were not significantly influenced by the absence of a specific questionnaire. Accordingly, we defined as potent those patients who had sexual intercourses with or without the aid of phosphodiesterase-5 blocking drugs. We believe that this also reduced the impact of the absence of a specific questionnaire regarding sexual potency. This is a pioneer study, as it comes from a private hospital in Brazil with no residence or fellow programs, analyzing a fairly recent technique that is not accessible to the majority of the patient and medical population. To our knowledge, this is also the first study reporting predictors of functional outcome recovery after RARP with more than one hundred patients.Age and obesity influenced the recovery of sexual potency, while only age was related to the recovery of urinary continence. We obtained good functional results, within the range of the largest published series, despite still within the learning curve. Prospective national studies with a larger number of patients are needed to better analyze functional results in larger Brazilian series.


Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) eggs (2 d), young larvae (7 d), old larvae (21 d), pupae (28 d), and adults (2 d after emergence) to 2, 4, 8, and 20.9% oxygen levels for 1, 3, 5, 10, and 15 d and assessed subsequent mortality. At 2% oxygen, complete mortality was achieved in 3 d for eggs and young larvae, 10 d for old larvae and pupae, and 15 d for adults. At 4% oxygen, 15 d were required to kill all eggs and old larvae but not the other insect life stages. At 8% oxygen after 15 d, complete mortality of any insect life stage was not observed; but even a relatively short exposure (1–3 d) caused significant developmental delays in immature insects. Our study shows potential utility of hermetic technology for control of T. castaneum, but internal oxygen should be maintained below 2% level for at least 15 d for complete control. Increased oxygen levels improved the development of all insect life stages leading to increased adult emergence. There is a need to explore exposure time required to achieve complete mortality of all insect life stage above the 2% oxygen level.Hermetic grain storage technology offers a viable chemical-free approach to control storage insects. However, there is limited knowledge on how hypoxia affects the survival of insect life stages during grain storage in hermetic bags. We exposed  Tribolium castaneum (Herbst) , the red flour beetle, is an important pest of stored cereal grains, pulses, and oilseeds. It has one of the highest population growth potentials for any stored-product insects   . Extensicticides . FurtherCallosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae), Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrichidae), Oryzaephilus surinamensis (Linnaeus) (Coleoptera: Silvanidae), Sitophilus oryzae (Linnaeus) (Coleoptera: Curculionidae), S. zeamais Motschulsky (Coleoptera: Curculionidae), and Trogoderma granarium Everts (Coleoptera: Dermestidae)  respiration of insects and other organisms present including the grain itself and 2) making the storage system airtight to greatly restrict the movement of air from outside . Studiesestidae) . In the T. castaneum eggs and adults  alone or in combination with temperatures  used by the Stored Product Insect Rearing Facility at the Department of Entomology, Purdue University, West Lafayette, IN. Colonies were reared in a CARON Insect Growth Chamber  at 27 ± 1°C, 60% relative humidity (RH), and at 16:8 (L:D) h photoperiod. The developmental life stages selected for the experiment were: egg (2 d), young larva (7 d), old larva (21 d), pupa (28 d), and freshly emerged unmated , and mixed-sex adults (1–2 d after emergence).T. castaneum . For each combination of oxygen level  and life stage treatments, 10 glass containers were prepared. We chose these low-oxygen levels  because they have been observed in hermetic bags during grain storage and have shown to preserve grain quality if the airtight conditions are maintained for several weeks.Clear glass containers (30 ml) with plastic lids containing approximately 0.5 g of wheat flour added as food for the insects were used for each hypoxia treatment . Lids were perforated with several small holes to allow gas exchange when placed in chambers at different oxygen levels. Each container received a single 3, 35 liters capacity  were used to expose insects to different levels of hypoxia. The experiment was conducted at room temperature (23°C). Each chamber received 10 glass containers for each of the life stages of T. castaneum (single insect in each container), and the remaining 10 containers for each life stage used as control were kept at ambient oxygen levels (20.9%). Three of the polycarbonate chambers were randomly chosen for given levels of hypoxia  using a random sequence generator . This was done because each treatment combination (hypoxia levels and exposure periods) had 10 vials each containing a single individual of the life stage. The 10 vials for each treatment were held inside the same hypoxia chamber and were considered as pseudoreplicates.n = 10). They were held in growth chambers  h photoperiod) for 10 d. After 10 d, postexposure survivorship was determined using the same assessment as discussed earlier.At the end of each hypoxia treatment, the adults were removed from the hypoxia chamber and examined immediately. They were classified as live if they were capable of reflex movement and dead if they did not move when prodded with a probe. Data were recorded as a binary response. If an adult was alive, it was given the value 1, and a value of 0 if dead. Controls were classified in the same manner. To assess the effect of hypoxia on adults beyond the initial survivorship, all adults for each treatment group were placed into a single vial with food  of adult emergence.Exposed eggs, young larvae, old larvae, and pupae were held for a maximum of 90 d posttreatment in the incubator  until any adult emergence had occurred. Adult emergence from exposed immatures of each treatment was recorded as a binary response, as earlier. Thus, if an adult emerged from an immature stage that had been exposed to hypoxia, it was recorded as 1. Similarly, if the immature stage failed to develop as an adult within 90 d, it was assigned a value of 0. In addition, the immature developmental time for Wald’s test was used to determine whether the covariance estimate of the random effect from three experimental runs was significantly different from 0 or not. If the experimental run showed no significant differences, data were pooled together for the variable experimental run for further analysis. To assess adult survivorship, logistic regressions were constructed using SAS PROC GLIMMIX Maximum Likelihood Estimation based on Laplace Approximation with the fixed effects of oxygen levels and exposure duration . For theP ≤ 0.05.Similarly, for the immature stages, logistic regression was constructed to model the proportion of adult emergence from immature stages using SAS PROC GLIMMIX Maximum Likelihood Estimation based on Laplace Approximation with the main effects of oxygen levels, exposure duration, and life stages. Data for the developmental period were analyzed using PROC GLM. Treatment means were separated using adjusted Tukey at There was no significant difference among temperatures and among RH for treatment kept inside and outside the hypoxia treatment chambers (data not shown). The mean and standard deviation of temperature for 20.9, 8, 4, and 2% oxygen levels were 24.01 ± 1.19, 23.69 ± 2.23, 23.56 ± 2.05, and 23.19 ± 1.66°C, respectively. Similarly, the mean and standard deviation of RH for 20.9, 8, 4, and 2% oxygen levels were 48.47 ± 8.61, 66.17 ± 15.69, 66.54 ± 12.54, and 61.96 ± 12.40, respectively.2 = 0.67, P = 0.21). Accordingly, the data were pooled together and analyzed for the effects of oxygen levels and exposure time. In the posttreatment assessment that begun immediately after returning insects to normoxia, we observed 100% adult survivals in control treatments and those exposed at all levels of hypoxia for 1 and 3 d. The data for 1 and 3 d and the controls were excluded from further analysis since there was no variation in the responses. We only used the hypoxia treatments of 2, 4, and 8% oxygen levels and for 5, 10, and 15 d to examine if there were posttreatment effects on adult survival. The GLIMMIX test of fixed effects showed that adult survival was significantly affected by 1) the oxygen levels , 2) exposure time , and 3) the interactions of oxygen levels × survival days . The probability of adult survival decreased as the oxygen levels decreased from 8 to 2% and likewise decreased as the length of exposure increased from 5 to 15 d. When exposed to 2% oxygen for 15 d, more than 99% of the exposed adults died. Exposure to 4% oxygen for more than 10 d resulted in more than half of the adults’ dead. In contrast, at 8% oxygen, over 90% of the insects survived even when exposed for 15 d , duration of exposure to hypoxia , and observation time after hypoxia treatment (d)   . About 3exposure .2 = 0.53, P = 0.23). The test of fixed effects revealed that adult emergence from exposed immatures was significantly affected by 1) the insect developmental life stage exposed to hypoxia , 2) oxygen levels , 3) exposure time , and 4) the interactions of life stages × oxygen levels × exposure time . Eggs were the most susceptible immature life stage to hypoxia treatment . However, to reach 90% mortality at 4% oxygen, adults required exposure for 15 d. On the other hand, exposure to 8% oxygen for 15 d produced <5% mortality. Our findings are consistent with those of previous researchers who reported that most stored product insects are sensitive to the oxygen levels <5%,  immediately after hypoxia treatments of low oxygen levels such as 2 or 4% were, in fact, moribund and died by 10 d posttreatment. The survival of T. castaneum adults 10 d after posttreatment appeared to decrease with increased duration of exposure to 2% oxygen, though there was no change to insects exposed for 1 and 15 d. Insect exposed to 2% oxygen for 1 d were all alive while those exposed for 15 d were all dead. Exposure to 2% for 3 d resulted in 30% posttreatment death 10 d later. Though all insects were alive immediately after 2% exposure for 3 d, T. castaneum adults were severely affected resulting in additional death 10 d later. Further investigations of insect behavior and physiology of the surviving adults would shed valuable light on the still largely unknown sublethal effects of hypoxia on insects.Further evidence of the sublethal effects of hypoxia is our present observations of additional mortality of T. castaneum respond differently to hypoxia. The egg was the most susceptible stage, with 100% mortality to 2% oxygen when exposed for at least 3 d. C. maculatus to 2% oxygen for 2 d suppressed adult emergence by over 80%, and increasing the exposure time to 3 d, reduced adult emergence by >98% compared with eggs exposed to normal ambient oxygen levels. Oxygen levels of 4% or less for 3 d or more killed all eggs. These findings corroborate results by T. castaneum eggs to be the most tolerant stages to conventional pest control methods such as grain fumigants used in crop storage protection systems  to 8% oxygen levels caused marked developmental delays in immature T. castaneum. Previously Tenebrio molitor L. (Coleoptera: Tenebrionidae) exposed to 10% oxygen levels molted 12.3 times compared to 5.8 times at normoxia, thereby supporting the assumptions of hypoxia-related developmental delay in insects. In this study, we found that exposure to 8% oxygen for 1 and 3 d increase development time by 21 and 43.5%, respectively, when compared exposure with normoxia. Exposure to 4% oxygen for 1 and 3 d increase the development time by 39.4 and 52.1%, respectively, when compared exposure with normoxia. This delay will have the effect of retarding population growth, thereby helping make the hermetic system more effective in controlling the numbers of insect present.The pupal stage was the least sensitive of the immature stages to hypoxia. This may be due to depressed respiratory metabolism during pupation . FurtherT. castaneum eggs, the most tolerant stage to the conventional fumigants, can be controlled using hermetic storage with oxygen level of 2% for at least 3 d; while other juvenile stages would require exposure to this same oxygen level for 10 d. Adults of T. castaneum require exposure period of 15 d at 2% oxygen level to produce complete mortality. However, hermetic containers with oxygen maintained at 4% would require 15 d to kill eggs and old larvae but not the other insect life stages. Oxygen levels of 8% may not directly contribute to the death of adults and immature stages of T. castaneum but can increase the developmental time of the immature stages, thereby slowing the rate of population expansion. Maintaining oxygen levels inside hermetic containers at 2% for at least 15 d will help control T. castaneum.In conclusion,


Illicit drug use is a growing public health problem. The aim of the study was to assess the prevalence of drug use and the sociodemographic and health characteristics that influence it among young and adult South Africans.Data based on the South African national population-based survey in 2012 for 26 453 individuals (52.0% women and 48.0% men) aged 15 years and older were analysed. Past 3-month drug use was assessed with the ‘Alcohol, Smoking and Substance use Involvement Screening Test (ASSIST)’. Bivariate and multivariable logistic regression was conducted to assess the association between sociodemographic factors, health variables and any past 3-month drug use.Overall, any past 3-month drug use was 4.4%, 7.9% among men and 1.3% among women. The proportion of past 3-month cannabis use was 4.0%, followed by sedatives or sleeping pills 0.4%, amphetamine-type stimulants 0.3%, cocaine 0.3%, opiates 0.3%, inhalants 0.2% and hallucinogens 0.1%. Among the nine South African provinces, any past 3-month drug use was the highest in the Western Cape (7.1%), followed by the Free State (6.3%) and Northern Cape (5.2%). In adjusted, multivariable, logistic regression analysis among both men and women, younger age, being mixed race and hazardous or harmful alcohol use were associated with any past 3-month drug use. In addition, having been a victim of violent crime and sexual risk behaviour among men and having psychological distress among women were associated with any past 3-month drug use.An increase of any past 3-month drug use from 3.7% in 2008 to 4.4% in 2012 was observed in South Africa. Prevention and intervention activities targeting drug use, in particular in identified risk groups, need to be strengthened in South Africa. The estimated global prevalence of illicit drug use  is 5.3% in the past year in 2014.4 In a South African population-based national study conducted in 2008, the prevalence of past 3-month (any) drug use was 3.7%.5 The highest past 3-month prevalence was for cannabis (3.3%), followed by sedatives or sleeping pills (0.8%), amphetamine-type stimulants (0.7%), cocaine (0.6%), opiates (0.5%) and hallucinogens (0.5%).5In a US population-based survey conducted among individuals aged 12 years and older in 2015, past 1-month (any) illicit drug use was 10.1%.6 younger age,5 specific population groups (mixed race and white people),6 lower income or not employed5 and geolocality such as urban areas.7 Further, certain health risk behaviours such as common mental disorders (major depression and anxiety disorders),9 alcohol use disorders,10 HIV risk behaviours11 and criminal victimisation12 have been found to be associated with drug use.Previous investigations in South Africa and other countries found that specific sociodemographic factors were associated with drug use, including male gender,In order to update planning on drug use programming, more recent national population-based prevalence data on illicit drug use among adolescents and adults in South Africa are needed. Therefore, the purpose of this secondary analysis was to make more recent estimates on the frequency and type of drug use among women and men 15 years and older available using a nationally representative household survey in 2012.13 The sampling strategy was stratified by province, type of geolocality and predominant population or racial groups. Using multistage sampling, a random sample of ‘enumeration areas’ was selected, and within enumeration areas households were randomly selected. All individuals within a household were eligible to participate. Trained and supervised field workers interview-administered a questionnaire. Informed consent was attained prior to the conduct of the interview. The detailed survey methods are described elsewhere.13Cross-sectional data from the ‘2012 South African national HIV prevalence, incidence, and behaviour survey’ was analysed.Drug use in the past 3 months was assessed with 10 items of the ‘Alcohol, Smoking and Substance Involvement Screening Test (ASSIST)’, e.g. ‘In the past 3 months, how often have you used cannabis ?’14 This included the item ‘Besides drugs prescribed by a health professional, have you ever used a drug by injection?’ The response option was ‘never’, ‘Yes. In the past 3 months’ and ‘Yes, but not in the past 3 months’. One item was added ‘Whoonga ’.13 Response options ranged from 1 = never to 5 = almost daily. Any drug use in the past 3 months was coded as 1 and never as 0. All items were added together to indicate the prevalence of any illicit drug use in the past 3 months. Cronbach’s alpha for this section of the ASSIST in this sample was 0.61.Demographic measures included age, educational level, race (African black people or other races), geolocality, province and employment status.Psychological distress was measured with the ‘Kessler Psychological Distress Scale (K-10)’.15 The 10-item scale assesses global psychological distress experienced in the past 30 days, e.g. ‘In the past 30 days, how often did you feel so restless that you could not sit still?’ Response options ranged from 1 = ‘none of the time’ to 5 = ‘all the time’. These scores were added up, with higher total scores indicating higher psychological distress.15 A cut-off of 16 scores and more for detecting depression and anxiety disorders was used, as suggested in a previous validation study of the K-10 in the general population in South Africa.16 Cronbach’s alpha for the K-10 in this sample was 0.89.Hazardous or harmful alcohol use was assessed with the 10-item ‘Alcohol Disorder Identification Test (AUDIT)’, e.g. ‘How often did you have a drink containing alcohol in the past 12 months?’17 Response options ranged from 0 to 4, with a summed total range from 0 to 40 scores; a score of 8 or more indicated hazardous or harmful or probable dependent drinking.17 Cronbach’s alpha for the AUDIT in this sample was 0.84.Violent crime victimisation was assessed with the question, ‘In the past 12 months, have you been a victim of a violent crime where a gun or knife was used to threaten or harm you?’ Response option was ‘yes’ or ‘no’.13Sexual risk behaviour was assessed with two questions. (1) ‘Have you had sex during the past 12 months?’ (2) ‘Overall, how many sexual partners did you have during the past 12 months?’ .13Whoonga), sociodemographic and health risk-independent variables were examined by calculating odds ratios. Unconditional multivariable logistic regression was utilised to assess the impact of explanatory variables for the outcome of past 3-month drug use prevalence, for women and men separately. All variables that were statistically significant at the p < 0.05 levels in bivariate analyses were included in the multivariable models. In the article, weighted percentages are presented. The ‘svy’ command was utilised to take into account the multistage cluster design of the survey. All statistical analyses were performed by using Stata software version 12 .Descriptive statistics were used to summarise drug use prevalence, sociodemographic factors and health variables. Associations between the outcome variable of any past 3-month drug use ’ and by the ‘Centers for Disease Control and Prevention’ (CDC).13 The total sample included 26 453 persons aged 15 years and older, 52.0% women and 48.0% men. More than half of the participants (52.0%) were between 15 and 34 years old, 40.2% had Grade 12 or more education, 77.8% were from the African black population group, 39.3% were formally or informally employed and 51.9% lived in urban formal areas. A large proportion of the participants (37.8%) reported psychological distress, 11.1% hazardous or harmful or probable dependent alcohol use, 14.7% had become victim of a violent crime in the past 12 months and 19.1% had more than one sexual partner in the past 12 months.Response rates for the interview was 89.5%.Whoonga , etc.)’ 0.3%, ‘inhalants ’ 0.2%, and ‘hallucinogens ’ 0.1%. The prevalence of past 3-month drug by injection was 0.6%, and the use of Whoonga 0.2%. Among the nine South African provinces, any past 3-month drug use was the highest in the Western Cape (7.1%), followed by the Free State (6.3%), Northern Cape (5.2%) and Gauteng (4.9%) ’ 0.4%, amphetamine-type stimulants  0.3%, ‘cocaine ’ 0.3%, ‘opiates  see .In adjusted, multivariable, logistic regression analysis among men, younger age, being mixed race, not living in a rural informal area, hazardous or harmful alcohol use, having been victim of a violent crime and having two or more sexual partners in the past 12 months were associated with any past 3-month drug use. In adjusted, multivariable, logistic regression analysis among women, younger age, being white people or of mixed race, not unemployed (looking for job), psychological distress and hazardous or harmful alcohol use were associated with any past 3-month drug use see .5 but lower than in a survey in the US (past month 10.1%).4 Among the different drugs used, the highest past 3-month prevalence was for cannabis (4.0%), as found in the 2008 South Africa survey (3.3%)5 and also in other countries such as the US.4 Compared to the 2008 South Africa national survey,5 in this 2012 survey the prevalence of past 3-month use increased from 3.3% to 4.0% for cannabis use, while it decreased for sedatives or sleeping pills from 0.8% to 0.4%, amphetamine-type stimulants 0.7% to 0.3%, cocaine 0.6% to 0.3%, opiates 0.5% to 0.3%, hallucinogens from 0.5% to 0.1% and inhalants from 0.5% to 0.2%.5 The increase in the prevalence of past 3-month drug use from 2008 to 2012 seems to be even higher among the youth (15–24 years) from 4.2% to 5.7%. This seems to show that the increase in any drug use is mainly attributed to an increase in cannabis use and a decrease in poly drug use (two or more drugs) from 0.7% in 2008 to 0.5% in 2012. The increase in the prevalence of cannabis use may be attributed to lower costs and better accessibility than the other drugs. One review18 indicates a global increase in substance use, including illicit drug use, in particular among young people, emphasising the importance of interventions. The use of Whoonga does not seem to be insignificant. In a qualitative study:In this very large national population-based study of 2012 among individuals 15 years or older found that any past 3-month drug use was 4.4%, which was higher than in the previous 2008 national survey, with 3.7%19perceptions of whoonga suggest that it is highly addictive, contains ARVs (notably efavirenz), and poses a threat to the health and safety of those who use it, including increasing the risk of HIV infection.tik and others had been as high as 2.2% in the Western Cape province in the 2008 survey,5 but was only 0.8% in the Western Cape in this survey, which may indicate some form of control as compared to previously.20 The increase in the use of drugs by injection from 0.3% in 2008 to 0.6% in 2012 is of concern. A study of 450 people who inject drugs (PWID) in South Africa found that they engaged in high HIV risk behaviour, including 49.0% having ‘used contaminated injecting equipment the last time they injected’.21The use of amphetamine-type stimulants including 22 including in South Africa, the prevalence of drug use was significantly higher among men than women. Further, in agreement with some previous studies,6 this study found that younger age and being from the mixed race population group were associated with drug use. Unemployment was among men in bivariate analysis associated with drug use, as found in some previous studies.5As found in previous studies,12 this study found that hazardous or harmful or probable dependent alcohol use among both men and women, having had more than one sexual partner in the past 12 months among men, having been victim of violent crime among men and psychological distress (anxiety and depression) among women were associated with drug use. Drug use prevention and intervention have to include co-morbidity factors such as common mental disorders, alcohol use disorders and sexual risk behaviour.In agreement with previous reviews,23 Furthermore, the internal consistency of the ASSIST questionnaire for the drug use section was only moderate (0.61). Possible reasons for this may be that alcohol and tobacco use were not assessed as part of the ASSIST and an additional item (Whoonga use) had been added. Further, the ASSIST assesses methadone as an opioid, while technically it is a synthetic opioid.This study was cross-sectional and no causative conclusions between independent variables and drug use can be drawn. The data on drug use were collected by self-report and may underreport the true consumption rate.An increase of any illicit drug use prevalence rates was observed from 2008 to 2012 in South Africa. Prevention and intervention activities targeting illicit drug use, in particular in identified risk groups, need to be strengthened in South Africa.


To analyze the safety and efficacy of high-myopic PRK as an alternative to LASIK surgery in patients with a high preoperative percentage tissue altered (PTA).  Retrospective interventional case series.  Charts of 256 consecutive eyes that underwent PRK with application of mitomycin-C 0.02% for high myopia were retrospectively reviewed.  Refractive (refraction and refractive accuracy) and visual outcomes , as well as occurrence of haze in the eyes with preoperative PTA expected to be higher than 40% with a 110-micron flap if undergoing LASIK surgery, were analyzed.  Mean follow-up was 7.3 ± 4.8 months. A total of 187 of 256 eyes (73.0%) were included in the analysis because they were expected to have a PTA greater than 40%, should they have undergone LASIK surgery. The actual mean PTA of those eyes following PRK was 31.8 ± 2.2%, and none had a PTA ≥ 40%. UDVA of 20/16, 20/20, and 20/25 or better was achieved in 1.2% (2 eyes), 65.5% (112 eyes), and 85.4% (146 eyes), respectively. The percentage of eyes with postoperative SE within ±0.5 D and ±1.0 D of planned SE was 71% and 93%, respectively. None of the eyes lost 2 or more lines of CDVA. The rate of stromal haze, managed successfully with topical steroids only, was 4.8%.  High-myopic PRK with application of mitomycin-C in the eyes at risk of developing ectasia because of high preoperative PTA was demonstrated to be a safe and effective alternative to the LASIK procedure. Excimer laser refractive surgery in patients with high myopia is associated with an increased complication rate. Laser-assisted in situ keratomileusis (LASIK) in cases of high myopia carries an increased risk of iatrogenic ectasia . RecentlExcimer laser technology has evolved continuously. Current generation laser platforms offer faster ablation rates, more sophisticated laser delivery methods and algorithms, optimized ablation profiles, and accurate eye tracking. Additionally, the routine application of mitomycin-C (MMC) has substantially decreased rates of stromal haze formation .Nevertheless, PRK in patients with high myopia remains a challenge for refractive surgeons. Several large series (larger than 100 eyes) previously examined PRK in high myopia, using older generation laser platforms. While it was found to be safe and effective, the PRK outcome was not comparable to the outcome of PRK in low to moderate myopia –14. In tThis retrospective study was performed at the Tel Aviv Medical Center, Israel, and was conducted in accordance with the tenets of the Declaration of Helsinki. We reviewed records of patients that underwent PRK for high myopia (greater than 6D) between January 2013 and July 2013 at Care-Vision Laser Centers, Tel Aviv, Israel. The data were routinely collected and entered into the electronic medical record database by the facility staff.All patients underwent a complete preoperative ophthalmological examination that included measurements of monocular uncorrected distance visual acuity (UDVA) and corrected distance visual acuity (CDVA), manifest and cycloplegic refraction, slit-lamp biomicroscopy, pupillometry, corneal topography, pachymetry, applanation tonometry, and dilated fundoscopy. Soft contact lenses and rigid gas-permeable contact lenses were removed at least 1 or 2 weeks, respectively, prior to the preoperative examination.The eyes were included in the analysis when they met the following criteria: the eyes that underwent PRK for high myopia greater than −6 D with at least 3 months of follow-up and normal preoperative topography, defined as regular and symmetric patterns  or mildly asymmetric  based on Placido disk analysis  and haviμm estimated epithelial depth)/preoperative corneal thickness] × 100.Percentage of tissue altered (PTA) has been previously described and defined for LASIK surgery as  × 100 . PTA wasThe calculation of the mean gain in visual acuity (VA) was made on the basis of VA values obtained with Snellen decimals, and these values were converted into LogMAR. The conversion of decimal VA values according to Snellen decimal into LogMAR was made by using the following formula: LogMAR = −log. The final expression of VA in the decimal value was reconverted with the following formula:−logMAR. The calculation of a variation of visual acuity in terms of the number of lines was established according to the formula [)–log] × 10. The quantitative values were expressed as mean and standard deviation.Decimal VA = 10Reasons for choosing PRK over LASIK in this group were young age and occupational reasons , pachymetry less than 500 microns or predicted LASIK RSB less than 300 microns.All the PRK procedures were performed by 6 different ophthalmic surgeons according to the same surgical protocol and with the wavefront-optimized photoablation profile (WFO) using the WaveLight EX500 excimer laser . The surgical procedures were performed with a 0.95 mm spot size, an optical zone of either 6.00 or 6.50 mm, and a transition zone of 1.25 mm. The magnitude of the spherical and astigmatic correction was first determined using the WaveLight EX500 platform's integrated nomogram, and adjustments were made by the surgeon based on the final assessment of all available clinical data.Epithelium removal was performed using 20% ethanol placed on the cornea in an 8.5 mm well for 15 seconds. The cornea was then rinsed with a balanced salt solution, and a blunt spatula was used to peel off the epithelium followed by stromal ablation using the excimer EX500 excimer laser. In all the eyes, a sponge soaked with 0.02% MMC was placed on the stroma for 30 to 90 seconds (according to the depth of the ablation) immediately after excimer ablation. After rinsing to remove the MMC, a contact lens was placed. Following surgery, moxifloxacin 0.5% 4 times daily for 1 week, dexamethasone 0.1% 4 times daily for 4 weeks followed by a taper, and preservative-free artificial tears for several months were prescribed. The eyes were examined for 1 day, 1 and 6 weeks, and 3 and 12 months postoperatively. UDVA, CDVA manifest refraction, and slit-lamp biomicroscopy were evaluated at every postoperative examination. Corneal topography was performed at 6 weeks, 3 months, and 12 months postoperatively.t-test and between subjects using the independent sample t-test. In cases of multiple independent comparisons, one way ANOVA was used.Data were recorded in Microsoft Excel and analyzed using SPSS version 21 . Postoperative data were collected at the 3rd month and at the last follow-up visit for the analysis. Continuous variables, such as visual acuity, were compared within subjects using the paired For small group comparison and ordinal variables, the Wilcoxon nonparametric test was used for paired comparisons and the Mann–Whitney nonparametric test was used for independent samples. Multiple independent comparisons were performed by the Kruskal-Wallis nonparametric test.p value <0.05.Binary variables were compared within subjects using the McNemar test for symmetry and between subjects using Fisher's exact test or Pearson chi-square test. To analyze the correlation between continuous variables, Pearson's correlation test was used. Univariate analysis and multivariate logistic regression models were constructed in order to test the association between multiple relevant covariates and haze formation. All tests were 2-tailed, and the threshold for statistical significance was defined as a This study included 256 highly myopic eyes of 129 subjects, and 106 eyes were eyes of female participants (41.4%). The mean follow-up time was 7.3 ± 4.8 months. Baseline clinical and demographic characteristics of the subjects are summarized in Of the 256 highly myopic eyes that underwent PRK, none had actual PRK PTA >40%. The average PTA was 30.5 ± 3.0% (range 22.9% to 38.0%). If those eyes would have had LASIK performed, 187 eyes (73.0%) would have had a PTA > 40% with a 110-micron flap and 134 eyes (52.3%) would have had a PTA > 40% with a 100-micron flap. Efficacy and safety analysis was performed for the patients with expected PTA > 40% in 110-micron flap LASIK. All 187 analyzed eyes had at least 3 months of follow-up, and 65 of 187 eyes were examined at the 12-month follow-up. Outcomes of the 3rd month and of the last available follow-up visit are presented (last follow-up time ranges from 3 to 20 months).Postoperative SE at 3 months and at the final follow-up was within ±0.5 D of the target SE in 70.4% and 71.0% of the eyes, respectively, and within ±1.00 D in 92.2% and 93.0% of the eyes, respectively. Refractive astigmatism at 3 months and at the final follow-up was within ±0.50 D in 77.1% and 72.0% of the eyes, respectively, and within ±1.00 D in 96.7% and 90.0% of the eyes, respectively. Of the 171 eyes corrected for distance, UCVA of 20/16, 20/20, 20/25, 20/40, or better at 3 months was achieved in 0 eyes (0.0%), 57 eyes (33.3%), 140 eyes (81.9%), and 168 eyes (98.2%), respectively. UCVA of 20/16, 20/20, 20/25, 20/40, or better at the final follow-up was achieved in 2 eyes (1.2%), 112 eyes (65.5%), 146 eyes (85.4%), and 170 eyes (99.4%), respectively. The efficacy index was 1.017.p < 0.001 for both) than preoperative CDVA .Postoperative CDVA at 3 months  and at the final follow-up  was significantly better (One eye (0.5%) gained 2 lines of CDVA. There were no eyes that lost 2 or more lines of CDVA. The safety index was 1.046.All procedures were uneventful. There were no major postoperative complications. Stromal haze was documented in 9 eyes of 6 patients (4.8% of 187 eyes). The degree of haze was grade 1 in 8/9 eyes and grade 3 in 1/9 eyes. Management in all cases consisted of a change in the topical steroidal regimen. None of the eyes required further intervention due to haze. There were no cases of corneal ectasia.There were no significant final follow-up outcome differences between the simulated PTA > 40% groups and the entire PRK cohort .In this study, we evaluated the efficacy and safety of PRK, performed with a new-generation excimer laser platform, in a large series of eyes with high myopia which would otherwise have a PTA > 40% and would be considered at high risk for ectasia if operated using LASIK.A total of 65.5% of the eyes corrected for distance had UCVA of 20/20 or better, and 99.4% had UCVA of 20/40 or better. The postoperative SE was within ±0.5 D of the target SE in 71% of the eyes and within ±1.00 D in 93% of the eyes. Several large series (more than 100 eyes in each) investigated PRK in high myopia –14. TheiWe were unable to find large cohorts of highly myopic eyes who underwent PRK in the recent literature. A recent study evaluated 77 eyes with high myopia that underwent either PRK with MMC or LASEK, both using the LADARVision 4000 excimer laser system . In this study, 84.4% of the eyes had postoperative UCVA of 20/20 or better . This maIn a recent study, femto-LASIK performed on 134 highly myopic eyes showed a 3-month predictability of 60.4% and 87.3% for SE within ±0.50 D and ±1.00 D, respectively. UCVA of 20/20 or better was documented in 82.8% of the patients, while preoperative CDVA of 20/20 or better was documented in 89.6% of the patients . WavefroWhile efficacy of LASIK may be slightly superior to that of PRK in patients with high myopia, the risk of ectasia may be substantially increased due to the deep stromal alteration. The value of PTA was found to be a robust indicator of the risk for post-LASIK ectasia, and a value of more than 40% is associated with increased ectasia risk . The aveThis study was aimed at showing early outcomes of high-myopic PRK as an alternative to LASIK in high-risk eyes. The risk for ectasia in high-myopic PRK patients should be evaluated in the long-term, although ectasia following PRK is extremely rare and has been recently shown to be 0.029% , with none of the ectasia cases being highly myopic . FurtherIn conclusion, current PRK in the eyes with high myopia is efficacious, accurate, and safe. In cases with high ectasia risk due to increased PTA, PRK is a solid alternative to LASIK.


Brucella field strains submitted for typing to the Italian National Reference Laboratory for Brucellosis from 2007 to 2015. Strains were identified at the species and biovar levels by classic and molecular techniques according to the World Organisation for Animal Health Manual. In total, 5,784 strains were typed: 3,089 Brucella abortus (53.4%), 2,497 B. melitensis (43.2%), 10 B. ovis (0.2%), 181 B. suis (3.1%), and 7 B. ceti (0.1%). The 2,981 strains from cattle were typed as B. abortus biovars 1, 3, and 6 (90.1%) and B. melitensis biovar 3 (9.9%). The 318 strains from water buffalo were typed as B. abortus biovars 1, 3 (95.9%) and B. melitensis biovar 3 (4.1%). The 2,279 strains from sheep and goats were typed as B. abortus biovars 1 and 3 (4.3%); B. melitensis biovars 1, 3, (95.3%); and B. ovis (0.4%). The 173 strains from wild boar were typed as B. suis biovar 2 (98.3%) and B. melitensis biovar 3 (1.7%). The 11 strains from pigs were typed as B. suis biovar 2. The 13 strains from humans were typed as B. melitensis biovar 3. The two strains from horses were typed as B. abortus biovar 1, while the seven strains from dolphins were typed as B. ceti. This additional knowledge on the epidemiology of brucellosis in Italy may be useful to formulate policies and strategies for the control and eradication of the disease in animal populations. The animal species affected, biovars typed, geographical origins, and spatial distributions of isolates are herein analyzed and discussed.Brucellosis is a major public health problem still prevalent as a neglected endemic zoonosis requiring proactive attention in many communities worldwide. The present study involved analysis of  Brucella. The disease may affect cattle, sheep, goats, pigs, and humans. Having a worldwide distribution, it is one of the most important zoonoses in the Mediterranean and Middle East regions. Eleven species are recognized within the genus  for species and biovar typing, according to the rules stated in the Ministerial Order of 14 November 2006. Specimens were transported and delivered in accordance with the World Health Organization (WHO) safety guidelines [www.izs.it). The Brucella polyvalent and monospecific Brucella A and M antisera were supplied by the Food and Agriculture Organization/WHO Collaborating Centre for Research on Brucellosis . All Brucella field isolates were subcultured in Brucella agar base and stored using the Microbank system  at −80°C.All strains were isolated by the local Italian Istituti Zooprofilattici Sperimentali (State Veterinary Laboratories) at idelines  and the idelines , 27. TheBrucella was performed by culturing the samples in Brucella broth supplemented with Farrell’s mix of antibiotics [Brucella agar  supplemented with 5% horse serum and antibiotics at the following amounts per 1 L of media: bacitracin (25 000 IU), polymyxin B (5000 IU), natamycin (50 mg), nalidixic acid (5 mg), nystatin (100 000 IU), and vancomycin (20 mg). The broth was incubated at 37°C ± 2°C in an atmosphere supplemented with 5% to 10% CO2 (v/v) for up to 6 weeks. From the broth, two plates per sample were inoculated each week: one plate was incubated in aerobiotic conditions at 37°C ± 2°C and the other in an atmosphere supplemented with 5 to 10% CO2 (v/v) at 37°C ± 2°C. The plates were observed after 3 days and then daily to identify Brucella-like colonies. The plates were discarded if no specific growth was evident after 7 to 10 days of incubation. Suspected colonies were subcultured onto serum dextrose agar from which subsequent growth was examined microscopically using Gram stain and biochemical  and motility tests.According to the technique described in the OIE Manual of Diagnostic Tests and Vaccines , 29, theibiotics  and on Babortus, melitensis, ovis, suis) polymerase chain reaction (PCR)  and PCR-restriction fragment length polymorphism techniques (PCR-RFLP). AMOS-PCR is a multiplex PCR designed to detect four species of Brucella [Brucella chromosome. Individual biovars within a species are not differentiated [Identification were performed with AMOS , and the products of digestion were checked by fluorescence after electrophoresis in 3% agarose gel in the presence of ethidium bromide. The specific biovar pattern was obtained by crossing the results of the single omp restrictions [Three different PCRs were used to amplify three outer membrane protein genes of rictions , 35, 36.2S; CO2-dependence; and growth in the presence of basic fuchsin and thionin at a final concentration of 20 μg/ml.For B.abortus and B.suis, to complete the biovars differentiation, addictional tests were performed agglutination with anti-A, anti-M, and anti-R monospecific sera; the production of HTable 1. Overall, 5,784 strains submitted from 13 regions in Italy were analyzed. Strains isolated from livestock were obtained from cattle , water buffalo , sheep , goats , pigs , wild boar , horses , and dolphins . Thirteen strains included in the study were isolated from humans. The strains isolated from cattle were B. abortus biovars 1, 3, and 6. Brucella melitensis biovar 3 was also isolated from cattle in several regions. In water buffalo, B. abortus biovars 1 and 3, and B. melitensis biovar 3 were identified. The strains isolated from sheep and goats were mainly B. melitensis biovar 3. B. melitensis biovar 1, and B. abortus biovar 1 and 3 were also isolated from these animal species. The only strain isolated from pigs was B. suis biovar 2. The same biovar was also isolated from wild boars. Brucella abortus biovar 1 was isolated from horses. Brucella ceti was isolated from some specimens of Stenella coeruleoalba found dead on the Italian coast. Infection was detected in 13 humans and was caused by B. melitensis biovar 3 in all cases. The relative percentage of isolation within animal species is shown in  The strains identified from cattle showed a high prevalence of B. abortus biovar 3 isolates (84.5%) followed by B. melitensis biovar 3 (9.9%) and B. abortus biovars 1 and 6   (. Some isolates of B. melitensis biovar 3 (4.1%) were also identified   and B.ovis (0.5%) (. The isolates from goats were B. melitensis biovar 3 (96.3%) and B. abortus biovar 3 (3.5%)  was identified. A total of 98.3% of isolates from wild boar were typed as B. suis biovar 2  . Several B. melitensis isolates were also recorded in the present study, both in cattle and water buffalo , documentation of this occurrence is lacking [A strain of ectively , 58, 59. lacking .B. ceti is common in cetaceans, but only small proportions of infected cetaceans display pathological signs associated with brucellosis, excluding the striped dolphin . This may suggest that many infected cetaceans overcome infection, perhaps remaining as carriers and potential Brucella shedders [B. ceti confirms the presence of the bacterium in the Tyrrhenian and Adriatic Seas. Although Brucella strains from land animals have not been identified in cetaceans, B. ceti strains have been isolated from human cases, stressing the zoonotic potential of this strain. However, despite the few human cases attributed to Brucella isolated from marine mammals, the magnitude of the risk that B. ceti represents for humans remains unknown.Infection by shedders . The recB. melitensis biovar 3, and all were identified in hospitals from the Lombardia, Piedmont, Campania, Emilia Romagna, Lazio, and Puglia regions. This is consistent with previous findings where, in Italy, brucellosis caused by B. melitensis was the most important clinically apparent disease recorded in humans. However, all human cases reported in the present study were notified in hospitals. Actually, because the Italian legislation requires human cases of brucellosis to be reported by the hospitals at which the cases are investigated, the site of disease notification is often different from the site at which the patient acquired the infection. In our cases, strains were received by some of hospitals for supporting the isolation or the typing process, and this may also explain the lower rate of isolation in humans than in animals.All isolates from human samples were S1 File(XLSX)Click here for additional data file.