Isolation and characterization of Leptospira licerasiae in Austrian swine - a first-time case report in Europe.

BACKGROUND: Leptospiraceae comprise a diverse family of spirochetal bacteria, of which many are involved in infectious diseases of animals and humans. Local leptospiral diversity in domestic animals is often poorly understood. Here we describe the incidental detection of Leptospira (L.) licerasiae in an Austrian pig. CASE PRESENTATION: During an experiment to characterize the pathogenesis of L. interrogans serovar Icterohaemorrhagiae in pigs, cultivation of a urine sample from a non-challenged contact pig resulted in growth of a spirochetal bacterium that tested negative for pathogenic Leptospira (LipL32 gene). PCR, Sanger sequencing and standard serotyping further confirmed that the recovered isolate was clearly different from the challenge strain L. interrogans serovar Icterohaemorrhagiae used in the animal experiment. Whole genome sequencing revealed that the isolate belongs to the species L. licerasiae, a tropical member of the Leptospiraceae, with no prior record of detection in Europe. CONCLUSIONS: This is the first report describing the occurrence of L. licerasiae in Europe. Since L. licerasiae is considered to have intermediate pathogenicity, it will be important to follow the geographical distribution of this species and its pathogenic and zoonotic potential in more detail.

Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic Leptospira.

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with Leptospira borgpetersenii serogroup Ballum (n = 48) and/or Leptospira kirschneri serogroup Icterohaemorrhagiae (n = 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them: L. borgpetersenii serogroup Ballum in LR1, LR5, and LR37, and L. kirschneri serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of Leptospira concurrently. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.IMPORTANCEPathogenic Leptospira, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of Leptospira may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic Leptospira from the same host. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.

Searching and Finding the Hidden Treasure: A Retrospective Analysis of Rickettsial Disease Among Dutch International Travelers.

BACKGROUND: Rickettsial disease (RD) is a prevalent and underestimated cause of febrile illness worldwide, especially in the absence of an inoculation eschar. We attempted to quantify this underestimation at our clinic, by investigating past cases of febrile illness in travelers who had tested negative for leptospirosis, a disease that can initially present similarly to non-eschar RD, and which we routinely consider when other important causes of unspecified febrile illness have tested negative. METHODS: We performed a retrospective analysis in febrile returned travelers from Asia, Africa, or the Americas between 2010 and 2017, who had tested negative for leptospirosis. Serologic immunofluorescence assays were performed for Orientia tsutsugamushi (scrub typhus), typhus group, and spotted fever group RD. We performed a medical records review of all patients who tested positive. In case of a fitting medical history, cases were deemed either confirmed (based on convalescent serology) or suspected (based on single serology). RESULTS: Among 97 patients, convalescent serology was available in 16 (16.5%) patients, and a single serology in 81 (83.5%) patients. RD was the likely diagnosis in 8 of 16 (50.0%) patients with convalescent serology, and in 8 of 81 (9.9%) with single serology. Of the 16 confirmed/suspected cases, 11 (69%) had been missed and 7 (44%) had not received adequate empiric antibiotic therapy. CONCLUSIONS: This study shows that non-eschar RD is an important and poorly recognized cause of illness in travelers, even in a specialized travel clinic. A lower threshold to test and treat for RD is warranted in returning travelers with febrile illness.

Safe Upper Limits of Serum Cobalt and Chromium Levels for a Metal-on-Metal Total Hip Bearing: A 10-Year Follow-Up Study.

BACKGROUND: Long-term survival of metal-on-metal (MoM) prostheses and the development of adverse reaction to metal debris (ARMD) around these bearings are still unclear. Serum levels of cobalt (Co) and chromium (Cr) are used as a screening tool to anticipate failure in MoM bearings and detect ARMD. METHODS: One hundred sixty primary large head MoM prostheses were followed up for 10 years. To estimate the revision risk, the cumulative incidence function (CIF) was used. Subdistribution hazard modeling was used to investigate the associations between cumulative incidence of revision for ARMD and Co levels, Cr levels, gender, age, head size, and cup inclination. Furthermore, the safe upper limits (SULs) for Co and Cr were determined. RESULTS: Univariate analyses showed an increased risk in revision for ARMD in females (subdistribution hazard ratio [sdHR] 3.43, 95% confidence interval [CI] 1.01-11.7, P = .049) and cup inclination angles over 45° (sdHR 4.70, 95% CI 1.63-13.58, P = .004). In addition, a higher last measured Co level (sdHR 1.05, 95% CI 1.03-1.07, P < .001) and last measured Cr level (sdHR 1.21, 95% CI 1.14-1.29, P < .001) were associated with a higher probability of revision for ARMD. We determined our bearing-specific SULs at 4.1 parts per billion (ppb) and 4.2 ppb for Co and Cr, respectively. CONCLUSION: Guidelines regarding follow-up and surveillance should include a complete clinical assessment with bearing-specific SULs of serum metal ion levels. For the M2a-Magnum MoM bearing we advise an SUL for Co and Cr levels of 4.1 and 4.2 ppb, respectively.

Introduction of primary screening using high-risk HPV DNA detection in the Dutch cervical cancer screening programme: a population-based cohort study.

BACKGROUND: In January 2017, the Dutch cervical cancer screening programme transitioned from cytomorphological to primary high-risk HPV (hrHPV) DNA screening, including the introduction of self-sampling, for women aged between 30 and 60 years. The Netherlands was the first country to switch to hrHPV screening at the national level. We investigated the health impact of this transition by comparing performance indicators from the new hrHPV-based programme with the previous cytology-based programme. METHODS: We obtained data from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for 454,573 women eligible for screening in 2017 who participated in the hrHPV-based programme between 1 January 2017 and 30 June 2018 (maximum follow-up of almost 21 months) and for 483,146 women eligible for screening in 2015 who participated in the cytology-based programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). We compared indicators of participation (participation rate), referral (screen positivity; referral rate) and detection (cervical intraepithelial neoplasia (CIN) detection; number of referrals per detected CIN lesion). RESULTS: Participation in the hrHPV-based programme was significantly lower than that in the cytology-based programme (61% vs 64%). Screen positivity and direct referral rates were significantly higher in the hrHPV-based programme (positivity rate: 5% vs 9%; referral rate: 1% vs 3%). CIN2+ detection increased from 11 to 14 per 1000 women screened. Overall, approximately 2.2 times more clinical irrelevant findings (i.e. ≤CIN1) were found in the hrHPV-based programme, compared with approximately 1·3 times more clinically relevant findings (i.e. CIN2+); this difference was mostly due to a national policy change recommending colposcopy, rather than observation, of hrHPV-positive, ASC-US/LSIL results in the hrHPV-based programme. CONCLUSIONS: This is the first time that comprehensive results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with a long follow-up. We have shown that both benefits and potential harms are higher in one screening round of a well-implemented hrHPV-based screening programme than in an established cytology-based programme. Lower participation in the new hrHPV programme may be due to factors such as invitation policy changes and the phased roll-out of the new programme. Our findings add further to evidence from trials and modelling studies on the effectiveness of hrHPV-based screening.

Colorectal metastasis to the gallbladder mimicking a primary gallbladder malignancy: histopathological and molecular characteristics.

AIMS: Outcomes of colorectal cancer (CRC) treatment and survival have steadily improved during the past decades, accompanied by an increased risk of developing second primary tumours and metastatic tumours at unusual sites. Metastatic CRC can show mucosal colonisation, thereby mimicking a second primary tumour. This potential confusion could lead to incorrect diagnosis and consequently inadequate treatment of the patient. The aim of this study was to differentiate between metastatic CRC and a second primary (gallbladder cancer, GBC) using a combination of standard histopathology and molecular techniques. METHODS AND RESULTS: Ten consecutive patients with both CRC and GBC were identified in our region using the Dutch National Pathology Archive (PALGA). Two patients served as negative controls. Histology of GBC was reviewed by nine pathologists. A combination of immunohistochemistry, microsatellite analysis, genomewide DNA copy number analysis and targeted somatic mutation analysis was used to aid in differential diagnosis. In two patients, CRC and GBC were clonally related, as confirmed by somatic mutation analysis. For one case, this was confirmed by genomewide DNA copy number analysis. However, in both cases, pathologists initially considered the GBC as a second primary tumour. CONCLUSIONS: Metastatic CRC displaying mucosal colonisation is often misinterpreted as a second primary tumour. A combination of traditional histopathology and molecular techniques improves this interpretation, and lowers the risk of inadequate treatment.

Concordance of Gleason grading with three-dimensional ultrasound systematic biopsy and biopsy core pre-embedding.

PURPOSE: To determine the value of a three-dimensional (3D) greyscale transrectal ultrasound (TRUS)-guided prostate biopsy system and biopsy core pre-embedding method on concordance between Gleason scores of needle biopsies and radical prostatectomy (RP) specimens. METHODS: Retrospective analysis of prostate biopsies and subsequent RP for PCa in the Jeroen Bosch Hospital, the Netherlands, from 2007 to 2016. Two cohorts were analysed: conventional 2D TRUS-guided biopsies and RP (2007-2013, n = 266) versus 3D TRUS-guided biopsies with pre-embedding (2013-2016, n = 129). The impact of 3D TRUS-guidance with pre-embedding on Gleason score (GS) concordance between biopsy and RP was evaluated using the κ-coefficient. Predictors of biopsy GS 6 upgrading were assessed using logistic regression models. RESULTS: Gleason concordance was comparable between the two cohorts with a κ = 0.44 for the 3D cohort, compared to κ = 0.42 for the 2D cohort. 3D TRUS-guidance with pre-embedding, did not significantly affect the risk of biopsy GS 6 upgrading in univariate and multivariate analysis. CONCLUSIONS: 3D TRUS-guidance with biopsy core pre-embedding did not improve Gleason concordance. Improved detection techniques are needed for recognition of low-grade disease upgrading.

Effectiveness of two strategies to follow-up ASC-US and LSIL screening results in The Netherlands using repeat cytology with or without additional hrHPV testing: a retrospective cohort study.

PURPOSE: The purpose of the study was to assess the effectiveness of repeat cytology with and without additional high-risk human papilloma virus (hrHPV) testing after atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion (ASC-US/LSIL) screening results. METHODS: In the Netherlands, ASC-US/LSIL is triaged by repeat cytology at 6 months or repeat cytology at 6 months with additional hrHPV testing. ASC-US/LSIL results from 13,734 screenees in 2008 were extracted from "Dutch Pathology Registry" including cytology, histology, and/or HPV follow-up results. Proportions of compliance, repeat cytology, referral, and detected cervical intraepithelial neoplasia (CIN) were assessed. RESULTS: With additional hrHPV testing, 46.8 % was send back to regular screening at 6 months, 28.6 % needed second repeat cytology, and 24.6 % was referred for colposcopy. Without additional hrHPV testing, this was 0.0, 76.1, and 23.9 %, respectively. With additional hrHPV testing, significantly higher proportions of persisting ASC-US/LSIL; compliance with repeat/referral advices; and histological detection of CIN0 (no CIN or cancer), CIN1, and CIN2 were found but equal proportions CIN3+. CONCLUSIONS: Additional hrHPV testing shortens follow-up without altering CIN3+ detection. Detection of CIN0, CIN1, and CIN2 was higher, presumably by hrHPV-driven biased cytology and detection bias. Restricting additional hrHPV testing to older women, reading cytology without knowledge of hrHPV status, and addition of more specific triage tests could further improve the effectiveness of additional hrHPV testing.

Is There an Added Value of Quantitative DCE-MRI by Magnetic Resonance Dispersion Imaging for Prostate Cancer Diagnosis?

In this multicenter, retrospective study, we evaluated the added value of magnetic resonance dispersion imaging (MRDI) to standard multiparametric MRI (mpMRI) for PCa detection. The study included 76 patients, including 51 with clinically significant prostate cancer (csPCa), who underwent radical prostatectomy and had an mpMRI including dynamic contrast-enhanced MRI. Two radiologists performed three separate randomized scorings based on mpMRI, MRDI and mpMRI+MRDI. Radical prostatectomy histopathology was used as the reference standard. Imaging and histopathology were both scored according to the Prostate Imaging-Reporting and Data System V2.0 sector map. Sensitivity and specificity for PCa detection were evaluated for mpMRI, MRDI and mpMRI+MRDI. Inter- and intra-observer variability for both radiologists was evaluated using Cohen's Kappa. On a per-patient level, sensitivity for csPCa for radiologist 1 (R1) for mpMRI, MRDI and mpMRI+MRDI was 0.94, 0.82 and 0.94, respectively. For the second radiologist (R2), these were 0.78, 0.94 and 0.96. R1 detected 4% additional csPCa cases using MRDI compared to mpMRI, and R2 detected 20% extra csPCa cases using MRDI. Inter-observer agreement was significant only for MRDI (Cohen's Kappa = 0.4250, p = 0.004). The results of this study show the potential of MRDI to improve inter-observer variability and the detection of csPCa.

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Aftercare following syndesmotic screw placement: a systematic review.

For ankle fractures, in general, several studies have been published on immobilization (e.g., cast or boot) versus early motion after surgical treatment. However, no studies have been performed to determine the best aftercare strategy for surgically treated patients with ankle fractures with concomitant acute distal tibiofibular syndesmotic injuries. The aim of the present review was to compare the functional outcomes of ankle fractures with syndesmotic injury treated with a cast or boot versus early motion. We performed a systematic review using the electronic databases from January 1, 2000 to September 1, 2012 of the Cochrane Library, PubMed MEDLINE(®), EMbase, and Google Scholar. The included studies were those in which ankle fractures with acute distal tibiofibular syndesmotic injuries had been treated with 1 or more syndesmotic screws, with a mean follow-up period of at least 12 months and at least 25 patients included. The functional outcomes, measured using the American Orthopaedic Foot Ankle Society Hindfoot scale, Olerud-Molander Ankle Scale, and Short Musculoskeletal Function Assessment, were compared. A total of 9 studies were identified with a total of 531 patients. The number of included patients ranged from 28 to 93. The mean follow-up period was 12 to 101 months. Of the 9 studies, 3 used an early motion protocol (195 patients) and 6 (336 patients) a protocol of immobilization for at least 6 weeks. For the American Orthopaedic Foot Ankle Society Hindfoot scale, the mean scores for immobilization were 86 to 91 points and for early motion, 84 to 89. For the Olerud-Molander Ankle Scale, the scores for immobilization were 47 to 90 and for early motion, 46 to 82 points. The Short Musculoskeletal Function Assessment score for immobilization was 11 and for early motion ranged from 12 to 27 points. No apparent differences could be detected in the published data considering the functional outcomes between immobilization versus an early motion protocol in ankle fractures with acute distal tibiofibular syndesmotic injuries treated with a syndesmotic screw. However, level 1 and 2 studies on this subject are lacking.

Reliability of whole mount radical prostatectomy histopathology as the ground truth for artificial intelligence assisted prostate imaging.

The development of artificial intelligence-based imaging techniques for prostate cancer (PCa) detection and diagnosis requires a reliable ground truth, which is generally based on histopathology from radical prostatectomy specimens. This study proposes a comprehensive protocol for the annotation of prostatectomy pathology slides. To evaluate the reliability of the protocol, interobserver variability was assessed between five pathologists, who annotated ten radical prostatectomy specimens consisting of 74 whole mount pathology slides. Interobserver variability was assessed for both the localization and grading of PCa. The results indicate excellent overall agreement on the localization of PCa (Gleason pattern ≥ 3) and clinically significant PCa (Gleason pattern ≥ 4), with Dice similarity coefficients (DSC) of 0.91 and 0.88, respectively. On a per-slide level, agreement for primary and secondary Gleason pattern was almost perfect and substantial, with Fleiss Kappa of .819 (95% CI .659-.980) and .726 (95% CI .573-.878), respectively. Agreement on International Society of Urological Pathology Grade Group was evaluated for the index lesions and showed agreement in 70% of cases, with a mean DSC of 0.92 for all index lesions. These findings show that a standardized protocol for prostatectomy pathology annotation provides reliable data on PCa localization and grading, with relatively high levels of interobserver agreement. More complicated tissue characterization, such as the presence of cribriform growth and intraductal carcinoma, remains a source of interobserver variability and should be treated with care when used in ground truth datasets.

Human leptospira isolates circulating in Mayotte (Indian Ocean) have unique serological and molecular features.

Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.

Bovine Leptospirosis Due to Persistent Renal Carriage of Leptospira borgpetersenii Serovar Tarassovi.

Leptospirosis is a global zoonotic disease that causes significant morbidity and mortality in human and animal populations. Leptospira interrogans is a leading cause of human disease, and L. borgpetersenii is a leading cause of animal disease. Cattle are reservoir hosts of L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges resulting in abortion and poor reproductive performance. Bovine bacterin vaccines can only protect against those serovars included in vaccine formulations and typically include serovar Hardjo among others. Genotyping and serotyping represent two different and unique methods for classifying leptospires that do not always correlate well; comprehensive characterization using either method requires recovery of isolates from infected animals. In this study, we report for the first time, isolation of L. borgpetersenii serovar Tarassovi from the urine of a dairy cow in the U.S. The classification of the isolate, designated strain MN900, was confirmed by whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, Matrix Assisted Laser Desorption/Ionization (MALDI), and immunoblotting with reference antisera. Strain MN900 was excreted in urine samples for 18 weeks even as the cow was seronegative for serovar Tarassovi. Strain MN900 has an unusual morphology since it is not as motile as other leptospires and lacks hooked ends. Serovar Tarassovi is not included in U.S. bacterin vaccines. These results demonstrate the importance of culture and concomitant genotyping and serotyping to accurately classify leptospires, and as required to design efficacious vaccine and diagnostic strategies to not only limit animal disease but reduce zoonotic risk.

Leptospira borgpetersenii serovar Hardjo and Leptospira santarosai serogroup Pyrogenes isolated from bovine dairy herds in Puerto Rico.

Leptospirosis is one of the most common zoonotic diseases in the world and endemic in the Caribbean Islands. Bovine leptospirosis is an important reproductive disease. Globally, cattle are recognized as a reservoir host for L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges, and can result in abortion and poor reproductive performance. The dairy industry in Puerto Rico comprises up to 25% of agriculture-related income and is historically the most financially important agricultural commodity on the island. In this study, we report the isolation of two different pathogenic Leptospira species, from two different serogroups, from urine samples collected from dairy cows in Puerto Rico: L. borgpetersenii serogroup Sejroe serovar Hardjo and L. santarosai serogroup Pyrogenes. Recovered isolates were classified using whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, and immunoblotting. These results demonstrate that dairy herds in Puerto Rico can be concurrently infected with more than one species and serovar of Leptospira, and that bacterin vaccines and serologic diagnostics should account for this when applying intervention and diagnostic strategies.

Genetic diversity of pathogenic leptospires from wild, domestic and captive host species in Portugal.

Leptospirosis is a neglected zoonotic disease of worldwide distribution with a significant veterinary and public health impact. It is caused by pathogenic bacteria of the genus Leptospira. The availability of effective tools to accurately identify and type leptospires is of utmost importance for the diagnosis of the disease and for assessing its epidemiology. Several multi-locus sequence typing (MLST) approaches were described for the typing of worldwide isolates of Leptospira but an extensive agreement towards the adoption of a unique consensus scheme for this agent is still lacking. Most genotyped strains originate from Asian and South American countries, with a minority originating from Europe (being most countries represented only by one or a few isolates). The knowledge of the diversity of circulating leptospires is the key to understanding the disease transmission and its zoonotic implications. In this study, we revisited the taxonomy of several isolates of pathogenic Leptospira obtained from domestic, wild and captive animals in Portugal, between 1990 and 2012. A selection of these isolates was genotyped using two previously published MLST schemes. A total of seven distinct sequence types (STs) were detected among the Portuguese isolates with two STs representing L. borgpetersenii (ST149 and ST152), two STs representing L. kirschneri (ST117 and ST100) and three STs representing L. interrogans (ST17, ST24 and ST140). Global widespread (and maybe more virulent) Leptospira genotypes seem to circulate in Portugal, particularly the L. interrogans ST17 isolates which are associated with several outbreaks of leptospirosis among humans and animals in different regions of the world. This study contributes to the enrichment of the global MLST databases with a new set of allele and sequence type information also providing novel data on circulating Leptospira serovars in Portugal.

Contrast-enhanced ultrasound tractography for 3D vascular imaging of the prostate.

Diffusion tensor tractography (DTT) enables visualization of fiber trajectories in soft tissue using magnetic resonance imaging. DTT exploits the anisotropic nature of water diffusion in fibrous structures to identify diffusion pathways by generating streamlines based on the principal diffusion vector. Anomalies in these pathways can be linked to neural deficits. In a different field, contrast-enhanced ultrasound is used to assess anomalies in blood flow with the aim of locating cancer-induced angiogenesis. Like water diffusion, blood flow and transport of contrast agents also shows a principal direction; however, this is now determined by the local vasculature. Here we show how the tractographic techniques developed for magnetic resonance imaging DTT can be translated to contrast-enhanced ultrasound, by first estimating contrast flow velocity fields from contrast-enhanced ultrasound acquisitions, and then applying tractography. We performed 4D in-vivo contrast-enhanced ultrasound of three human prostates, proving the feasibility of the proposed approach with clinically acquired datasets. By comparing the results to histopathology after prostate resection, we observed qualitative agreement between the contrast flow tracts and typical markers of cancer angiogenic microvasculature: higher densities and tortuous geometries in tumor areas. The method can be used in-vivo using a standard contrast-enhanced ultrasound protocol, opening up new possibilities in the area of vascular characterization for cancer diagnostics.

Prostate Cancer Risk Assessment in Biopsy-naïve Patients: The Rotterdam Prostate Cancer Risk Calculator in Multiparametric Magnetic Resonance Imaging-Transrectal Ultrasound (TRUS) Fusion Biopsy and Systematic TRUS Biopsy.

BACKGROUND: The value of multiparametric magnetic resonance imaging (mpMRI) and targeted biopsy (TBx) remains controversial for biopsy-naïve men when compared to transrectal ultrasound (TRUS)-guided systematic biopsy (SBx). Risk-based patient selection could help to selectively identify men with significant prostate cancer (PCa) and thus reduce unnecessary mpMRI and biopsies. OBJECTIVES: To compare PCa detection rates for mpMRI TBx with SBx and to determine the rate of potentially avoided mpMRI and biopsies through risk-based selection using the Rotterdam Prostate Cancer Risk Calculator (RPCRC). DESIGN, SETTING, AND PARTICIPANTS: Two-hundred consecutive biopsy-naïve men in two centres underwent mpMRI scanning, 12-core SBx, and subsequent MRI-TRUS TBx in the case of suspicious lesion(s) (Prostate Imaging-Reporting and Data System v.2 score ≥3). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We measured the detection rate for high-grade (Gleason score ≥ 3+4) PCa for TBx and SBx. We carried out a retrospective stratification according to RPCRC biopsy advice to determine the rate of mpMRI and biopsies that could potentially be avoided by RPCRC-based patient selection in relation to the rate of high-grade PCa missed. RESULTS AND LIMITATIONS: TBx yielded high-grade PCa in 51 men (26%) and low-grade PCa in 14 men (7%), while SBx yielded high-grade PCa in 63 men (32%) and low-grade PCa in 41 men (21%). Four out of 73 men (5%) with negative RPCRC advice and 63 out of 127 men (50%) with positive advice had high-grade PCa. Upfront RPCRC-based patient selection for mpMRI and TBx would have avoided 73 out of 200 (37%) mpMRI scans, missing two out of 51 (4%) high-grade PCas. Limitations include the RPCRC definition of high- and low-grade PCa and different mpMRI techniques. CONCLUSIONS: mpMRI with TBx detected PCa with high Gleason score and avoided biopsy in low-grade PCa, but failed to detect all high-grade PCa when compared to SBx among biopsy-naïve men. Risk-based patient selection using the RPCRC can avoid one-third of mpMRI scans and SBx in biopsy-naïve men. PATIENT SUMMARY: Men with a suspicion of prostate cancer are increasingly undergoing a magnetic resonance imaging (MRI) scan. Although promising, MRI-targeted biopsy is not accurate enough to safely replace systematic prostate biopsy for now. Individualised assessment of prostate cancer risk using the Rotterdam Prostate Cancer Risk Calculator could avoid one-third of MRI scans and systematic prostate biopsies.

Single nucleotide polymorphism profiling assay to confirm the identity of human tissues.

To identify issues of sample mix-ups, various molecular techniques are currently used. These techniques, however, are time consuming and require experience and/or DNA sequencing equipment or have a relatively high risk of errors because of contamination. Therefore, a quick and straightforward single nucleotide polymorphism (SNP) profiling assay was developed to link human tissues to a source. SNPs are common sequence variations in the human genome, and each individual has a unique combination of these nucleotide variations. Using potentially mislabeled paraffin-embedded tissues, DNA was extracted and SNP profiles were determined by real-time polymerase chain reaction analysis of the purified DNA using a selection of 10 commercially available SNP amplification assays. These profiles were compared with profiles of the supposed owners. All issues (34 in total) of potential sample mix-ups during the last 3 years were adequately solved, with six cases described here. The SNP profiling assay provides a quick (within 24 hours), easy, and reliable way to link human samples to a source, without polymerase chain reaction postprocessing. The chance for two randomly chosen individuals to have an identical profile is 1 in 18,000. Solving potential sample mix-ups will secure downstream evaluations and critical decisions concerning the patients involved.