Emission of extensively-drug-resistant Acinetobacter baumannii from hospital settings to the natural environment.

BACKGROUND: Acinetobacter baumannii is a leading emerging pathogen that is frequently recovered from patients during hospital outbreaks. The role of environmental A. baumannii reservoirs is therefore of great concern worldwide. AIM: To investigate the connection between A. baumannii causing hospital outbreaks and environmental isolates from hospital wastewater, urban sewage and river water as the final natural recipient of wastewaters. METHODS: Clinical isolates from patients with hospital-acquired pneumonia and environmental isolates from water were collected during a two-month monitoring period. Recovery of A. baumannii was performed using CHROMagar Acinetobacter plates, incubated at 42°C for 48 h. Identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and analyses of rpoB gene. The antibiotic resistance profiles were interpreted according to criteria given for clinical isolates of A. baumannii. The sequence types (ST) were retrieved by multi-locus sequence typing. RESULTS: Fourteen of 19 isolates recovered from patients, hospital wastewaters, urban sewage and river water belonged to ST-195. The remaining five isolates recovered from patients and river water were assigned to ST-1421. All isolates showed very strong relatedness and clustered into CC92, which corresponds to IC2. All isolates were non-susceptible to at least one agent in all but two or fewer antimicrobial categories, and thus were classified as 'extensively-drug-resistant' (XDR). Heteroresistance to colistin was found in two isolates from hospital wastewater. CONCLUSION: Close relatedness of clinical and environmental isolates suggests the emission of XDR A. baumannii via the untreated hospital wastewater in the natural environment.

A new laboratory model using bull and boar spermatozoa and fluorescent beads to assess a membrane's occlusive potential.

OBJECTIVES: The objective of the present study is to assess the potential of bull and boar spermatozoa and fluorescent beads to be used as a surrogate cell model to determine the cell occlusive potential in vitro using membranes of standardized porosities. MATERIALS AND METHODS: A two-chamber model system consisting of upper and lower chambers, which could be separated by membranes, was constructed. Isopore polycarbonate membranes with different standardized pore diameters were used to assess the mobile cellular penetration behavior of spermatozoa or the more passive non-cellular permeability of fluorescent particles (beads) of different diameter and color. In a first experiment, spermatozoa were placed in the lower chamber, whereas semen extender only was placed in the upper chamber. After 10 min of incubation at 37 °C, the sperm number was assessed in the latter. In a second experiment, a bead solution was drawn through resorbable collagen membranes from the upper into the lower chamber by vacuum using a syringe and bead number and size was analyzed by flow cytometry. All experiments were carried out in triplicates. A non-porous polyester membrane was used as negative control to assess the overall tightness of the setup. RESULTS: Boar and bull spermatozoa had average cell body lengths and widths of 9 × 5 µm and were unable to pass through pores ≤2 µm, whereas they were detectable at pore sizes ≥3 µm. Their number increased with increasing pore diameters, i.e., from minimal concentrations of 0.1 × 106/ml for boar and 0.5 × 106/ml for bull spermatozoa at 3 µm to maximal concentrations of 2.1 × 106/ml for boar and 13.1 × 106/ml for bull spermatozoa at 8 µm. The fluorescent beads followed the expected pattern of permeability reliably correlating bead and pore diameter. CONCLUSIONS: Within the limitations of this laboratory study and the xenogeneic cell surrogate material, the model allows to easily assess cell and particle penetration through porous structures like membranes. We hope to further assess, improve, and validate this model, which we aim to use for the screening of dental membranes after being exposed to different degradation methods. CLINICAL RELEVANCE: Convenient and rapid test procedures to evaluate membranes for regenerative procedures are still warranted.

Multilocus sequence analysis of 'Candidatus Phytoplasma asteris' strain and the genome analysis of Turnip mosaic virus co-infecting oilseed rape.

AIM: Molecular characterization of a pathogenic complex infecting winter oilseed rape (Brassica napus ssp. oleifera (DC.) Metzg.) plants showing typical rape phyllody symptoms along with some atypical changes. METHODS AND RESULTS: Phytoplasma ('Candidatus Phytoplasma') presence was confirmed by PCR-RFLP and 16S rRNA gene sequencing. Phylogenetic analyses of phytoplasma amp, tufB, secY, groEL and ribosomal protein genes confirmed its affiliation to the 'Ca. P. asteris' species. However, in the amp gene encoding a specific protein crucial for insect transmission specificity, significant SNPs were found. Biological and serological tests revealed the co-infection with Turnip mosaic virus (TuMV). The phylogenetic analysis of full TuMV genome sequence, the first reported from the Balkans, classified it into the world-B phylogenetic lineage. CONCLUSIONS: A pathogenic complex consisting of 'Ca. P. asteris' and TuMV found to co-infect oilseed rape plants for the first time was molecularly characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: Rape phyllody is a serious problem in rapeseed production. The molecular information from this first multi-gene analysis of 'Ca. P. asteris' strain associated with rape phyllody as well as the first report of the complete sequence of TuMV isolate from the Balkans is a starting point for understanding the disease complexity and management.

Electrochemotherapy: a new technological approach in treatment of metastases in the liver.

Electrochemotherapy is now in development for treatment of deep-seated tumors, like in bones and internal organs, such as liver. The technology is available with a newly developed electric pulse generator and long needle electrodes; however the procedures for the treatment are not standardized yet. In order to describe the treatment procedure, including treatment planning, within the ongoing clinical study, a case of successful treatment of a solitary metastasis in the liver of colorectal cancer is presented. The procedure was performed intraoperatively by inserting long needle electrodes, two in the center of the tumor and four around the tumor into the normal tissue. The insertion of electrodes proved to be feasible and was done according to the treatment plan, prepared by numerical modeling. After intravenous bolus injection of bleomycin the tumor was exposed to electric pulses. The delivery of the electric pulses did not interfere with functioning of the heart, since the pulses were synchronized with electrocardiogram in order to be delivered outside the vulnerable period of the ventricles. Also the post treatment period was uneventful without side effects. Re-operation of the treated metastasis demonstrated feasibility of the reoperation, without secondary effects of electrochemotherapy on normal tissue. Good antitumor effectiveness with complete tumor destruction was confirmed with histological analysis. The patient is disease-free 16 months after the procedure. In conclusion, treatment procedure for electrochemotherapy proved to be a feasible technological approach for treatment of liver metastasis. Due to the absence of the side effects and the first complete destruction of the treated tumor, treatment procedure for electrochemotherapy seems to be a safe method for treatment of liver metastases with good treatment effectiveness even in difficult-to-reach locations.

First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia.

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable. References: (1) L. Filippin et al. Plant Pathol. 58:826, 2009. (2) S. Kuzmanovic et al. Vitis 47:105, 2008. (3) C. Pelletier et al. Vitis 48:87, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Pre-operative ultrasound with a 12-15 MHz linear probe reliably differentiates between melanoma thicker and thinner than 1 mm.

BACKGROUND: Pre-operative determination of primary melanoma thickness could be a tool to identify those patients who could be treated with radical primary tumour excision and sentinel lymph node biopsy in a single procedure. An excellent correlation between sonographic and histological measurement of maximal tumour thickness has been achieved using 20-MHz transducers. OBJECTIVE: To show that widely available high resolution ultrasound with 12-15 MHz linear probe could also reliably assess the thickness of primary melanoma. METHODS: Sixty-nine patients underwent ultrasound evaluation of 70 clinically and dermoscopically suspicious pigmented skin lesions before surgical excision. RESULTS: The sensitivity, specificity, positive and negative predictive values of ultrasound to detect melanoma > 1 mm were 92%, 92%, 95% and 81% respectively. The correlation between ultrasound and histological tumour thickness was very good [Pearson's correlating index 0.823 (P < 0.001)]. Mean difference between sonographic and histological measurements was 0.045 mm with limits of agreement estimated at -1.4 and +1.49, and a bias between two methods 45 microm. CONCLUSION: Ultrasound examination with a 12-15 MHz linear transducer can reliably differentiate primary melanoma > 1 mm from those

Haematomas after percutaneus vacuum-assisted breast biopsy.

PURPOSE: Clinically apparent haematomas are among most frequent complications after vacuum-assisted breast biopsy (VABB). We evaluated the prevalence and persistence of sonographically (US) detected haematomas and other tissue changes at the biopsy site after VABB. MATERIALS AND METHODS: We examined 48 women who underwent stereotactic 11G needle VABB; the majority of them had mammographically detected microcalcifications. US examination of the breast biopsy site was performed one week after the VABB in 48 patients, and in 45 patients once again three weeks after the VABB. In 13/45 patients US-guided fine needle aspiration biopsy (FNAB) of the changes visualised was performed 3 weeks after the biopsy. RESULTS: One week after the VABB, a haematoma at the biopsy site was detected in 45/48 (94 %) patients (mean length 16.3 mm, mean width 3.6 mm). Three weeks after the VABB, haematoma was detected in 25/45 patients (55 %) (mean length 9.3 mm, mean width 2.7 mm), and architectural distortion in 13/45 patients (29 %), in 7/45 patients (16 %), no changes were found. In 13 patients in whom FNAB (fine needle aspiration biopsy) was performed, haematoma was found in 6/13, fat necrosis in 3/13, reactive changes in 2/13, whereas 2/13 samples were unsatisfactory. CONCLUSION: The changes at the biopsy site can be seen by US in most of the patients one and three weeks after the VABB. These changes could potentially be used for US guidance and localisation of microcalcifications in patients requiring surgical biopsy.

Identification of exhumed remains of fire tragedy victims using conventional methods and autosomal/Y-chromosomal short tandem repeat DNA profiling.

In a fire tragedy in Manila in December 1998, one of the worst tragic incidents which resulted in the reported death of 23 children, identity could not be established initially resulting in the burial of still unidentified bodies. Underscoring the importance of identifying each of the human remains, the bodies were exhumed 3 months after the tragedy. We describe here our work, which was the first national case handled by local laboratories wherein conventional and molecular-based techniques were successfully applied in forensic identification. The study reports analysis of DNA obtained from skeletal remains exposed to conditions of burning, burial, and exhumation. DNA typing methods using autosomal and Y-chromosomal short tandem repeat (Y-STR) markers reinforced postmortem examinations using conventional identification techniques. The strategy resulted in the identification of 18 out of the 21 human remains analyzed, overcoming challenges encountered due to the absence of established procedures for the recovery of mass disaster remains. There was incomplete antemortem information to match the postmortem data obtained from the remains of 3 female child victims. Two victims were readily identified due to the availability of antemortem tissues. In the absence of this biologic material, parentage testing was performed using reference blood samples collected from parents and relatives. Data on patrilineal lineage based on common Y-STR haplotypes augmented autosomal DNA typing, particularly in deficiency cases.

Allele frequencies of 19 STR loci in a Philippine population generated using AmpFlSTR multiplex and ALF singleplex systems.

Allele frequencies for the 19 short tandem repeat (STR) loci CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S306, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, DHFRP2 (FOLP23), F13A01, FES/FPS, FGA, TH01, TPOX, and vWA were obtained from a sample of 106 unrelated Filipinos from different regions of the Philippine archipelago.