Definition of clinically insignificant residual fragments after percutaneous nephrolithotomy among urologists: a world-wide survey by EAU-YAU Endourology and Urolithiasis Working Group.

Introduction: The aim of this article was to evaluate the current perception of urologists as to what size is considered as a clinically insignificant residual fragment (CIRF). Material and methods: A survey was globally distributed to the members of the Endourological Society via SurveyMonkey. Results: A total of 385 participants responded to the survey on CIRF. Most participants considered 2 mm (29%) as CIRF threshold, followed by 3 mm (24%), 4 mm (22%), 0 mm (14%), 5 mm (8%) and 1 mm (3%). North American urologists considered CIRF to be smaller than urologists from Asia, Eurasia and South America, (p-values ≤0.001, 0.037 and 0.015 respectively). European urologists identified smaller CIRF in comparison to Asian urologists (p-value = 0.001). Urologists mainly using a pneumatic lithotripter accepted larger fragments as CIRF, compared to urologists mainly using ultrasonic devices or a combination of ultrasonic and pneumatic devices (p-value = 0.026 and 0.005 respectively). Similarly, urologists mainly performing X-Ray and ultrasound as post-operative imaging accepted larger fragments as CIRF in comparison to urologists mainly performing non-contrast computed tomography (p-value = 0.001). Conclusions: What is considered as CIRF varies between urologist from different continents and seems to be associated with the lithotripter used and the post-operative imaging modality of preference to assess treatment success.

Early bone ingrowth and segmental stability of a trussed titanium cage versus a polyether ether ketone cage in an ovine lumbar interbody fusion model.

BACKGROUND CONTEXT: Lumbar interbody fusion is an effective treatment for unstable spinal segments. However, the time needed to establish a solid bony interbody fusion between the two vertebrae may be longer than twelve months after surgery. During this time window, the instrumented spinal segment is assumed to be at increased risk for instability related complications such as cage migration or subsidence. It is hypothesized that the design of new interbody cages that enable direct osseointegration of the cage at the vertebral endplates, without requiring full bony fusion between the two vertebral endplates, might shorten the time window that the instrumented spinal segment is susceptible to failure. PURPOSE: To quantify the bone ingrowth and resulting segmental stability during consolidation of lumbar interbody fusion using two different cage types. STUDY DESIGN: Preclinical ovine model. METHODS: Seven skeletally mature sheep underwent bi-segmental lumbar interbody fusion surgery with one conventional polyether ether ketone (PEEK) cage, and one newly developed trussed titanium (TT) cage. After a postoperative time period of 13 weeks, non-destructive range of motion testing, and histologic analysis was performed. Additionally, sample specific finite element (FE) analysis was performed to predict the stability of the interbody fusion region alone. RESULTS: Physiological movement of complete spinal motion segments did not reveal significant differences between the segments operated with PEEK and TT cages. The onset of creeping substitution within the cage seemed to be sooner for PEEK cages, which led to significantly higher bone volume over total volume (BV/TV) compared with the TT cages. TT cages showed significantly more direct bone to implant contact (BIC). Although the mean stability of the interbody fusion region alone was not statistically different between the PEEK and TT cages, the variation within the cage types illustrated an all-or-nothing response for the PEEK cages while a more gradual increase in stability was found for the TT cages. CONCLUSIONS: Spinal segments operated with conventional PEEK cages were not different from those operated with newly developed TT cages in terms of segmental stability but did show a different mechanism of bone ingrowth and attachment. Based on the differences in development of bony fusion, we hypothesize that TT cages might facilitate increased early segmental stability by direct osseointegration of the cage at the vertebral endplates without requiring complete bony bridging through the cage. CLINICAL SIGNIFICANCE: Interbody cage type affects the consolidation process of spinal interbody fusion. Whether different consolidation processes of spinal interbody fusion result in clinically significant differences requires further investigation.

Combined effect of undersized surgical technique and axial compression on the primary implant stability and host bone architecture.

AIM: The aim of this study was to investigate the combined effect of the lateral-compression of host-bone (undersized-osteotomy-preparation) and axial-compression of host-bone (not drilling the full length of the implant) on the primary-implant-stability and the host-bone-architecture. MATERIALS AND METHODS: In this experimental-study, 44 dental implants (diameter-4.2 mm; length-10 mm; Dyna®) were installed in the femoral-condyles of four cadaver-goats using four different surgical approaches (11 implant/surgical approach; n = 11). Approach-1: Standard preparation according to the manufacturer's guidelines. The bone-cavity was prepared up to 10 mm in depth and 4 mm in diameter. Approach-2: Preparation up to 8 mm in depth and 4 mm in diameter. Approach-3: Preparation up to 10 mm in depth. Approach-4: The bone-cavity was prepared up to 8 mm in depth and 3.6 mm in diameter. Insertion torque (n = 11), removal torque (n = 7) and % bone-implant contact (n = 4) measurements were recorded. Bone architecture was assessed by micro-computer tomography and histological analysis (n = 4). RESULTS: For approaches 2, 3, and 4 (P < .05), insertion-torque values were significantly higher as compared to approach 1. Regarding the bone-implant-contact percentage (%BIC), approach 3 and 4 were significantly higher compared to approach 1 and 2 (P<.05). For approach 2, the %bone volume (%BV) was significantly higher as compared to approach 1 (P<.05) for the most the inner zone of host bone in proximity of the implant. CONCLUSION: Lateral and axial compression improved the primary-implant-stability and therefore this new surgical-technique should be considered as an alternative approach especially for placing implants in low-density bone. Nevertheless, additional in vivo studies should be performed.

Characteristics of current digital single-use flexible ureteroscopes versus their reusable counterparts: an in-vitro comparative analysis.

BACKGROUND: Single-use flexible ureterorenoscopes (fURSs) have been recently introduced aiming to offer solutions to the sterilization, fragility and cost issues of the reusable fURSs. In order to be a viable alternative, the single-use scopes must prove similar capabilities when compared to their reusable counterparts. The goal of our in-vitro study was to compare the current reusable and single-use digital fURSs regarding their deflection, irrigation and vision characteristics. METHODS: We compared in-vitro 4 single-use fURSs-LithoVue™ (Boston Scientific, Marlborough, Massachusetts, USA), Uscope™ (Zhuhai Pusen Medical Technology Co. Ltd., Zhuhai, Guangdong Province, China), NeoFlex™ (NeoScope Inc, San Jose, California, USA) and ShaoGang™ (YouCare Technology Co. Ltd., Wuhan, China) versus 4 reusable fURSs-FLEX-Xc (Karl Storz SE & Co KG, Tuttlingen, Germany), URF-V2 (Olympus, Shinjuku, Tokyo, Japan), COBRA vision and BOA vision (Richard Wolf GmbH, Knittlingen, Germany). Deflection and irrigation abilities were evaluated with different instruments inserted through the working channel: laser fibres (200/273/365 µm), retrieval baskets (1.5/1.9/2.2 Fr), guide wires [polytetrafluoroethylene (PTFE) 0.038 inch, nitinol 0.035 inch] and a biopsy forceps. A scoring system was designed to compare the deflection impairment. Saline at different heights (40/80 cm) was used for irrigation. The flow was measured with the tip of the fURS initially straight and then fully deflected. The vision characteristics were evaluated (field of view, depth of field, image resolution, distortion and colour representation) using specific target models. RESULTS: Overall, the single-use fURSs had superior in-vitro deflection abilities than the reusable fURSs, in most settings. The highest score was achieved by NeoFlex™ and the lowest by ShaoGang™. PTFE guide wire had most impact on deflection for all fURSs. The 200 µm laser fibre had the lowest impact on deflection for the single-use fURSs. The 1.5 Fr basket caused the least deflection impairment on reusable fURSs. At the end of the tests, deflection loss was noted in most of the single-use fURSs, while none of the reusable fURSs presented deflection impairment. ShaoGang™ had the highest irrigation flow. Increasing the size of the instruments occupying the working channel led to decrease of irrigation flow in all fURSs. The impact of maximal deflection on irrigation flow was very low for all fURSs. When instruments were occupying the working channel, the single-use fURSs had slightly better in-vitro irrigation flow than the reusable fURSs. The field of view was comparable for all fURSs, with LithoVue™ showing a slight advantage. Depth of field and colour reproducibility were almost similar for all fURSs. ShaoGang™ and Uscope™ had the lowest resolution. FLEX Xc had the highest image distortion while LithoVue™ had the lowest. Partial field of view impairment was not for Uscope™ and ShaoGang™. CONCLUSIONS: In-vitro, there are differences in technical characteristics of fURSs. It appears that single-use fURSs deflect better than their reusable counterparts. Irrespective of deflection, the irrigation flow of the single-use fURSs was slightly superior to the flow of the reusable fURSs. Overall, reusable fURSs had better vision characteristics than single-use fURSs. Further in-vivo studies might be necessary to confirm these findings.

Three-dimensional evaluation of marginal and internal fit of 3D-printed interim restorations fabricated on different finish line designs.

PurposeTo evaluate the influence of fabrication method and finish line design on marginal and internal fit of full-coverage interim restorations. MethodsFour typodont models of maxillary central-incisor were prepared for full-coverage restorations. Four groups were defined; knife-edge (KE), chamfer (C), rounded-shoulder (RS), rounded-shoulder with bevel (RSB). All preparations were digitally scanned. A total of 80 restorations were fabricated; 20 per group (SLA/3D-printed n=10, milled n=10). All restorations were positioned on the master die and scanned using micro-computed tomography. The mean gaps were measured digitally (ImageJ). The results were compared using MANOVA (α=.05). ResultsInternal and marginal gaps were significantly influenced by fabrication method (P=.000) and finish-line design (P=.000). 3D-Printed restorations showed statistically significant lower mean gap compared to milled restorations at all points (P=.000). The mean internal gap for 3D-printed restorations were 66, 149, 130, 95µm and for milled restorations were 89, 177, 185, 154µm for KE, C, RS, RSB respectively. The mean absolute marginal discrepancy in 3D-printed restorations were (30, 41, 30, 28µm) and in milled restorations were (56, 54, 52, 38µm) for KE, C, RS, RSB respectively. ConclusionsThe fabrication methods showed more of an influence on the fit compared to the effect of the finish-line design in both milled and printed restorations. SLA-printed interim restorations exhibit lower marginal and internal gap than milled restorations. Nonetheless, for both techniques, all values were within the reported values for CAD/CAM restorations. Significance3D-printing can offer an alternative fabrication method comparable to those of milled restorations.

Osteogenesis around CaP-coated titanium implants visualized using 3D histology and micro-computed tomography.

Calcium phosphate (CaP) coatings can enhance the performance of bone implants in compromised conditions, such as osteoporosis. Therefore, this study compared non-coated vs. CaP-coated (n = 8) titanium implants in osteoporotic ovariectomized (OVX) rats. Bone volume (BV) was assessed using micro-computer tomography (micro-CT) and three-dimensional (3D) histology, in three zones from the implant surface. Bone remodeling was assessed using fluorochrome labels and osteoclast staining. Micro-CT and 3D histology showed a BV reduction in OVX animals, of respectively 22.4 and 10.5%. BV was significantly increased inside all zones around CaP coatings, especially in the inner zone of the OVX animals. Fluorochrome labels were predominantly seen when the coating was applied. Osteoclasts were mainly found in the area remote from the surface of non-coated implants in control animals. For the coated implants, osteoclasts were distributed evenly, and present in direct vicinity of the surface. In conclusion, 3D histology is a suitable technique to obtain data and insight into bone architecture around implants at relatively high resolution. Bone formation was significantly reduced in osteoporotic animals. CaP coatings resulted in a higher BV directly around implants installed in osteoporotic animals, enhanced turnover, and a shift of remodeling activity toward the implant surface.

Injectable self-gelling composites for bone tissue engineering based on gellan gum hydrogel enriched with different bioglasses.

Hydrogels of biocompatible calcium-crosslinkable polysaccharide gellan gum (GG) were enriched with bioglass particles to enhance (i) mineralization with calcium phosphate (CaP); (ii) antibacterial properties and (iii) growth of bone-forming cells for future bone regeneration applications. Three bioglasses were compared, namely one calcium-rich and one calcium-poor preparation both produced by a sol-gel technique (hereafter referred to as A2 and S2, respectively) and one preparation of composition close to that of the commonly used 45S5 type (hereafter referred to as NBG). Incubation in SBF for 7 d, 14 d and 21 d caused apatite formation in bioglass-containing but not in bioglass-free samples, as confirmed by FTIR, XRD, SEM, ICP-OES, and measurements of dry mass, i.e. mass attributable to polymer and mineral and not water. Mechanical testing revealed an increase in compressive modulus in samples containing S2 and NBG but not A2. Antibacterial testing using biofilm-forming meticillin-resistant staphylococcus aureus (MRSA) showed markedly higher antibacterial activity of samples containing A2 and S2 than samples containing NBG and bioglass-free samples. Cell biological characterization using rat mesenchymal stem cells (rMSCs) revealed a stimulatory effect of NBG on rMSC differentiation. The addition of bioglass thus promotes GG mineralizability and, depending on bioglass type, antibacterial properties and rMSC differentiation.

Integrating asthma hazard characterization methods for consumer products.

Despite extensive study, definitive conclusions regarding the relationship between asthma and consumer products remain elusive. Uncertainties reflect the multi-faceted nature of asthma (i.e., contributions of immunologic and non-immunologic mechanisms). Many substances used in consumer products are associated with occupational asthma or asthma-like syndromes. However, risk assessment methods do not adequately predict the potential for consumer product exposures to trigger asthma and related syndromes under lower-level end-user conditions. A decision tree system is required to characterize asthma and respiratory-related hazards associated with consumer products. A system can be built to incorporate the best features of existing guidance, frameworks, and models using a weight-of-evidence (WoE) approach. With this goal in mind, we have evaluated chemical hazard characterization methods for asthma and asthma-like responses. Despite the wealth of information available, current hazard characterization methods do not definitively identify whether a particular ingredient will cause or exacerbate asthma, asthma-like responses, or sensitization of the respiratory tract at lower levels associated with consumer product use. Effective use of hierarchical lines of evidence relies on consideration of the relevance and potency of assays, organization of assays by mode of action, and better assay validation. It is anticipated that the analysis of existing methods will support the development of a refined WoE approach.

Resolution, sensitivity, and in vivo application of high-resolution computed tomography for titanium-coated polymethyl methacrylate (PMMA) dental implants.

OBJECTIVES: The aims of this study were (i) to determine the spatial resolution and sensitivity of micro- versus nano-computed tomography (CT) techniques and (ii) to validate micro- versus nano-CT in a dog dental implant model, comparative to histological analysis. MATERIAL AND METHODS: To determine spatial resolution and sensitivity, standardized reference samples containing standardized nano- and microspheres were prepared in polymer and ceramic matrices. Thereafter, 10 titanium-coated polymer dental implants (3.2 mm in Ø by 4 mm in length) were placed in the mandible of Beagle dogs. Both micro- and nano-CT, as well as histological analyses, were performed. RESULTS: The reference samples confirmed the high resolution of the nano-CT system, which was capable of revealing sub-micron structures embedded in radiodense matrices. The dog implantation study and subsequent statistical analysis showed equal values for bone area and bone-implant contact measurements between micro-CT and histology. However, because of the limited sample size and field of view, nano-CT was not rendering reliable data representative of the entire bone-implant specimen. CONCLUSIONS: Micro-CT analysis is an efficient tool to quantitate bone healing parameters at the bone-implant interface, especially when using titanium-coated PMMA implants. Nano-CT is not suitable for such quantification, but reveals complementary morphological information rivaling histology, yet with the advantage of a 3D visualization.

Osseointegration of oral implants after delayed placement in rabbits: a microcomputed tomography and histomorphometric study.

PURPOSE: This study compared osseointegration of implants placed 14 days after implant site preparation with that of immediately placed implants in rabbit femurs. MATERIALS AND METHODS: Implants were placed bilaterally in the femoral condyles of 12 rabbits. On one side, the implants were placed 14 days after osteotomy, and the other side received implants immediately after osteotomy. Healing was assessed by microcomputed tomography and histomorphometry. RESULTS: The delayed implants (placed 14 days after osteotomy) showed better osseointegration than the immediately placed implants. Bone-to-implant contact and bone volume, as assessed by histomorphometry and microcomputed tomography, were significantly higher for the implants placed after 14 days. CONCLUSIONS: From this study, it can be concluded that early osteotomy bed preparation and placement of implants after a 2-week delay predisposes to better bone-implant interface healing.

Bone formation analysis: effect of quantification procedures on the study outcome.

Quantification of the amount of newly formed bone is an essential part of bone regeneration studies. Histomorphometry, based on histological sections of plastic-embedded specimens, is the most frequently applied technique in this assessment. Before performing image analysis, a specific region of interest (ROI) has to be determined. Based on the histological procedure, different areas within the ROI can be discriminated and assigned to relevant tissue structures. However, in literature not much attention is paid to the effect of the histological procedures on the final outcome of the histomorphometrical measurements on bone regeneration. In this study, the histomorphometrical bone formation of the intramedullary cavity of the guinea pig tibia, filled with calcium phosphate cement, was quantified in plastic-embedded and paraffin-embedded specimens and in specimens analyzed with scanning electron microscopy in the backscattering mode (SEM-BS). The data showed that the histological procedure significantly affected the measured bone amount. Therefore, it is recommended that scaffold characteristics are carefully considered in selecting a proper technique for the analysis of bone formation in bone tissue engineering studies. The results of this study identified high-resolution SEM-BS and elastic van Gieson staining of decalcified histological sections as recommendable techniques for evaluating bone formation.

Scanning electron microscopy stereoimaging for three-dimensional visualization and analysis of cells in tissue-engineered constructs: technical note.

In tissue engineering research, various three-dimensional (3D) techniques are available to study cell morphology, biomaterials, and their relations. To overcome disadvantages of frequently used imaging techniques, in the current study stereoimaging scanning electron microscopy (SEM) is proposed. First, the 3D SEM application was validated using a series of standardized microspheres. Thereafter, MC-3T3 cell morphology was visualized and cell parameters as cell height were quantified on titanium and calcium-phosphate materials using 3D reconstruction software. Besides 3D visualization of the cells, quantitative assessment showed significant substrate dependency of cell spreading in time. Such quantification of cell spreading kinetics can be used for optimization of tissue engineering scaffold surface properties. However, further standardization of SEM image acquisition and 3D SEM software settings are still essential for 3D cell analysis.

Three-dimensional localization of implanted biomaterials in anatomical and histological specimens using combined X-ray computed tomography and three-dimensional surface reconstruction: a technical note.

For adequate histological processing of implanted biomaterials or tissue-engineered constructs, it is sometimes essential to obtain insight into the localization of structures inside the tissue samples. Observation of three-dimensional (3D) surface reconstruction, including basic photorealistic texture characteristics as surface pattern and color combined with X-ray computed tomography 3D reconstruction at different levels, is a useful approach to localize anatomical or implanted structures within experimental tissue samples. Because of the possible observation of structures of interest in a 3D environment, fusion of these techniques can greatly facilitate histological processing.

Micro-computed tomographical imaging of soft biological materials using contrast techniques.

The aim of this work was to introduce high-resolution computed tomography (micro-CT) for scaffolds made from soft natural biomaterials, and to compare these data with the conventional techniques scanning electron microscopy and light microscopy. Collagen-based scaffolds were used as examples. Unlike mineralized tissues, collagen scaffolds do not provide enough X-ray attenuation for micro-CT imaging. Therefore, various metal-based contrast agents were applied and evaluated using two structurally distinct scaffolds, one with round pores and one with unidirectional lamellae. The optimal contrast techniques for obtaining high-resolution three-dimensional images were either a combination of osmium tetroxide and uranyl acetate, or a combination of uranyl acetate and lead citrate. The data obtained by micro-CT analysis were in line with data obtained by light and electron microscopy. However, small structures (less than a few mum) could not be visualized due to limitation of the spot size of the micro-CT apparatus. In conclusion, reliable three-dimensional images of scaffolds prepared from soft natural biomaterials can be obtained using appropriate contrast protocols. This extends the use of micro-CT analysis to soft materials, such as protein-based biomaterials.

Evaluation of bone response to titanium-coated polymethyl methacrylate resin (PMMA) implants by X-ray tomography.

High-resolution three-dimensional data about the bone response to oral implants can be obtained by using microfocus computer tomography. However, a disadvantage is that metallic implants cause streaking artifacts due to scattering of X-rays, which prevents an accurate evaluation of the interfacial bone-to-implant contact. It has been suggested that the use of thin titanium coatings deposited on polymeric implants can offer an alternative option for analyzing bone contact using micro-CT imaging. Consequently, the aim of the current study was to investigate bone behavior to titanium-coated polymethylmethacrylate (PMMA) implants by micro-CT and histological evaluation. For the experiment titanium-coated PMMA implants were used. The implants had a machined threaded appearance and were provided with a 400-500 nm thick titanium coating. The implants were inserted in the right or left tibia of 10 goats. After an implantation period of 12 weeks the implants were retrieved and prepared for micro-computer tomography (microCT), light microscopy, and X-ray microanalysis. The micro-CT showed that the screw-threads and typical implant configuration were well maintained through the installation procedure. Overall, histological responses showed that the titanium-coated implants were well tolerated and caused no atypical tissue response. In addition, the bone was seen in direct contact with the titanium-coated layer. The X-ray microanalysis results confirmed the light microscopical data. In conclusion, the obtained results proof the final use of titanium-coated PMMA implants for evaluation of the bone-implant response using microCT. However, this study also confirms that for a proper analysis of the bone-implant interface the additional use of microscopical techniques is still required.

Intracellular localization of ornithine decarboxylase and its regulatory protein, antizyme-1.

The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocytoplasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function.

Automated identification of diploid reference cells in cervical smears using image analysis.

BACKGROUND: Acquisition of DNA ploidy histograms by image analysis may yield important information regarding the behavior of premalignant cervical lesions. Accurate selection of nuclei for DNA measurement is an important prerequisite for obtaining reliable data. Traditionally, manual selection of nuclei of diagnostic and reference cells is performed by an experienced cytotechnologist. In the present study, a method for the fully automated identification of nuclei of diploid epithelial reference cells in Feulgen- restained Papanicolaou (PAP) smears is described. METHODS: The automated procedure consists of a decision tree implemented on the measurement device, containing nodes with feature threshold values and multivariate discriminant functions. Nodes were constructed to recognize debris and inflammatory cells, as well as diploid and nondiploid epithelial cells of the uterine cervix. Evaluation of the classifier was performed by comparing resulting diploid integrated optical densities with those from manually selected reference cells. RESULTS AND CONCLUSION: On average, automatically acquired values deviated 2.4% from manually acquired values, indicating that the method described in this paper may be useful in cytometric practice.

Hantavirus pulmonary syndrome in Panama: identification of novel hantaviruses and their likely reservoirs.

Hantavirus pulmonary syndrome (HPS), a severe respiratory disease with high mortality caused by rodent-borne hantaviruses, has previously been identified in the United States and Canada as well as central and southern South America. In late 1999 and early 2000, an outbreak of acute illness compatible with HPS was reported in Los Santos, Panama, with the death of 3 of the 12 (25%) suspected cases. Hantavirus-specific antibodies were detected in patient sera, and virus RNA was detected by reverse transcriptase-polymerase chain reaction. Sequence analysis of virus genome N-, G1-, and G2-encoding fragments showed this to be a novel hantavirus, Choclo virus. Serologic and virus genetic analyses of rodents trapped in the area showed Oligoryzomys fulvescens to be the likely reservoir for the HPS-associated Choclo virus. In addition, Zygodontomys brevicauda rodents were shown to harbor another genetically unique hantavirus, Calabazo virus.