Technological advances in minimally invasive radiofrequency ablation of cardiac tissues

Ablative techniques have been sought in many circumstances as alternatives to surgical resection/incision. Besides being minimally invasive, potential benefits of ablation include greater speed and improved access to target tissue compared with other surgical techniques. There is a wide variety of ablation technologies currently in use for medical treatment. These include but are not limited to tissue heating by radiofrequency (RF) current, microwaves, laser, and high intensity ultrasound. RF is among the most heavily used because of its relatively low complexity and cost. Ablative techniques have proven to be viable alternatives to surgical resection/incision of tissue. Although there are other means of tissue heating besides RF, RF is the most commonly used technique in operating rooms because of the reliability of transmural lesions and the low complexity of the system. Optimal systems account for the heterogeneous nature of tissue and variations in tissue property through the ablation cycle. It is important to monitor and assure adequate energy delivery by selecting the appropriate configuration of devices. Energy delivery varies between the various generators and systems, some more responsive than others with relative to changes in tissue impedance that will affect the end results of the operation.     

病毒分子生物学诊断方法的应用进展

作为病毒诊断实验室必备的一种手段 ,PCR相关的病毒分子生物学诊断技术准确、快速、敏感、简便 ,对指导临床治疗十分重要。本文对近年来 PCR技术方法学研究与应用研究及生物传感器诊断病毒等方面的一些进展进行了综述     

Myiasis caused by Oestridae: serological and molecular diagnosis

Myiasis-causing Oestridae (bot flies) infect several animal species world-wide, from palaearctic to subtropical/tropical areas. Oestrids affect livestock production causing abortion, reduced milk production, losses in weight and fertility, poor hide quality and an impairment of the host's immune system. In the last few years much research has been carried out on the immunology of these infestations, in order to acquire efficient and reliable diagnostic serological tools; the genome of the different species of Oestridae has been studied to further their molecular identification, taxonomy and phylogenesis. The immunodiagnostic methods for many myiasis causing Oestrids have proven to be a viable alternative to the clinical parasitological examination or the post-mortem examination. Numerous serological tests have been developed for the diagnosis of bovine hypodermosis caused by Hypoderma bovis and Hypoderma lineatum, and ELISAs using larval hypodermin C as the antigen are currently used on serum, individual and pooled milk samples to detect the presence of circulating anti-Hypoderma antibodies. In Italy the best period to sample the animals is November-January, since it is in this period that the antibody kinetics of the animals reaches a peak. Recently the efficacy of the ELISA test on pasteurized milk samples has been demonstrated, allowing the diagnosis of bovine hypodermosis also in areas where there is no information on the presence of the disease and the sampling of the animals is laborious. The cross-reactivity between Przhevalskiana silenus antigens and anti-Hypoderma antibodies led to assessing the usefulness of a simple and cost-effective ELISA for the diagnosis of goat warble fly infection. In particular, it has been demonstrated that infected goats display an antibody peak in November-December in blood and milk, thus making this period suitable for sampling. Although no extensive data is available on the immunology of sheep and goat oestrosis caused by Oestrus ovis, the efficacy of ELISA has been demonstrated by correlating serological results with clinical post-mortem examinations. No immunological techniques are currently used to diagnose gasterophilosis of equids and only one study reports the efficacy of ELISA for detecting anti-Gasterophilus antibodies in infected equids. Several studies have been conducted into the molecular characterization of the mitochondrial DNA (mtDNA)--in particular of the gene encoding for the cytochrome oxidase I (COI)--for many free-living and parasitic arthropods for diagnostic, taxonomic and phylogenetic purposes. As regards Oestridae causing myiasis, the first study features a PCR-RFLP assay of the most common Italian species (i.e. H. bovis, H. lineatum, Gasterophilus intestinalis, P. silenus, O. ovis), which showed clear genetic differences among the genera examined, but no inter-specific variation between the two species of Hypoderma considered. The molecular characterization of the most variable region of the COI gene (encoding for the region from E4 to the terminal COOH) was able to clearly differentiate H. bovis and H. lineatum. The E4-COOH region of the COI gene has been characterized for 18 oestrid species and from a taxonomical point of view, molecular data confirm the morphological classification, with the examined species divided into four subfamilies. New insights have also been gained on the molecular differentiation of the most common species of Hypoderma (i.e. H. bovis, H. lineatum, Hypoderma actaeon, Hypoderma diana and Hypoderma tarandi) and, in particular, the restriction enzyme BfaI, provides a diagnostic profile that can be used to simultaneously differentiate all the species examined. The characterization of the E4-COOH COI gene and the hypervariable region of the gene encoding for the ribosomal Isu revealed the identity of Hypoderma sinense as a new species, infecting cattle and yaks in China. Finally, the molecular analysis of the same mitochondrial and ribosomal regions showed that P. silenus, Przhevalskiana aegagri and Przhevalskiana crossii are morphotypes of the same species.     

Comparative characterization of methanotrophic enrichments by serological and molecular methods

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A, were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches reveals more reliable information on the diversity of methanotrophs.     

Monitoring Ceratomyxa shasta infection during a hatchery rearing cycle: comparison of molecular, serological and histological methods

The prevalence of Ceratomyxa shasta infection in production stocks of steelhead Oncorhynchus mykiss and cutthroat trout O. clarki was monitored using a parasite-specific polymerase chain reaction (PCR) assay. For all 4 stocks of fish followed through their 1 yr rearing cycle, C. shasta infection was detected despite their genetic resistance to the disease and the treatment of the incoming water with ozone. Infection was confirmed using serological methods and standard histological procedures, except when prevalence was low (<10%). This suggests that at the lowest infection levels PCR is more sensitive than other methodologies, and can be used as an early indicator of infection. Results of the PCR assay continued to correlate with histological and serological detection as the numbers of parasites and the lesion severity increased over the rearing cycle. For both steelhead and cutthroat trout, early infections were characterized by large numbers of parasites on the epithelial surface, but with little associated inflammation. At release as yearlings, the infection prevalence in all stocks was greater than 90 % and the inflammatory response in many fish was extensive, with tissue necrosis and mucosal damage. Although C. shasta infections no longer result in high mortality at this facility, results of this study indicate that the parasite remains a contributor to low condition indices in these fish, despite their genetic resistance and ozone disinfection of the water supply.     

Automated knot tying for fixation in minimally invasive, robot-assisted cardiac surgery

Cardiovascular disease (CVD) is perhaps the most significant worldwide health issue. While open-heart surgery remains the predominant treatment, significant advancements have been made in minimally invasive surgery (MIS) and minimally invasive robot-assisted (MIRA) surgery. MIRA techniques offer many advantages over open-heart procedures and have extended the capabilities of MIS. However, these benefits come at the cost of increased operating times due to time spent tying knots. The additional bypass time limits patient access and is the most significant barrier to the adoption of MIRA techniques. This research seeks to overcome this barrier by designing a device for MIRA cardiac procedures that automates the knotting of sutures. If this task can be automated while ensuring the delivery of high-quality knots, great progress can be made in transforming the field. MIRA cardiac procedures can move from novel procedures performed by a select group of surgeons on a limited pool of patients to a viable alternative available to the majority of patients with CVD. In this research we propose a design for a self-contained device that delivers a locking knot. Results suggest that consistent knots can be delivered at a time savings of 12.5% and 26.4% over manual knots for trained and untrained users of a surgical robot, respectively.     

Comparative characterization of cultured methane-oxidizing bacteria by serological and molecular methods

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches reveals more reliable information on the diversity of methanotrophs.     

A completely detachable aortic clamping instrument for minimally invasive cardiac surgery

A minimally invasive cardiac surgery is becoming more popular and is still undergoing a refinement of surgical techniques and dedicated instrumentarium as well. New specifically designed instruments are quintessence of safe surgery with improving operative outcomes and comfortable operator-oriented working conditions. In this article, we attempt to present our early clinical experience with a new aortic clamping instrument specifically developed for limited single-access minimally invasive valve surgery.     

侵袭性真菌深部感染的早期实验室诊断

目的探讨假丝酵母菌甘露聚糖抗原和假丝酵母菌IgG/IgM抗体、曲霉半乳甘露聚糖抗原和烟曲霉IgG抗体在侵袭性真菌病早期临床诊断中的应用价值。方法收集已确诊侵袭性假丝酵母菌病患者18例,侵袭性烟曲霉病患者6例,单纯细菌感染患者20例,浅部真菌感染患者20例,健康体检者(正常对照组)20例,通过酶联免疫吸附法(ELISA)检测患者血清甘露聚糖和假丝酵母菌IgG/IgM抗体以及曲霉半乳甘露聚糖抗原和烟曲霉IgG抗体浓度,计算各指标的灵敏度、特异度、阳性预测值、阴性预测值和受试者工作特征(ROC)曲线下面积。结果甘露聚糖抗原和假丝酵母菌IgG/IgM抗体联合测定的敏感度为66.7%,特异度为83.3%,阴性预测值为100.0%,阳性预测值为85.7%,ROC曲线下面积为0.992(95%CI:0.974~1.000);半乳甘露聚糖抗原和烟曲霉IgG抗体联合测定的敏感度为66.7%,特异度为95.0%,阴性预测值为98.2%,阳性预测值为100.0%,ROC曲线下面积为0.978(95%CI:0.934~1.000)。结论甘露聚糖抗原和假丝酵母菌IgG/IgM抗体、半乳甘露聚糖抗原和烟曲霉IgG抗体联合检测对深部真菌感染的早期诊断具有重要意义。     

Evaluation of two methods for minimally invasive peripheral body temperature measurement in birds

Body temperature (T_b) is a valuable parameter when assessing the physiological state of animals, but its widespread measurement is often constrained by methods that are invasive or require frequent recapture of animals. Alternatives based on automated remote sensing of peripheral T_b show promise, but little is known about their strengths and limitations. We measured peripheral T_b in great tits Parus major with subcutaneously implanted passive integrated transponders (PIT tags) and externally attached radio transmitters to determine repeatability of measurements, sensitivity of each method to variation in ambient temperature (T_a) and wind speed, the relationship between methods, and their ability to capture circadian variation in T_b. Repeatability of measurements by radio transmitters was high (> 80%) when readings were taken within 20 min, but reduced to 16% when measures were spaced 3.5 h apart. PIT tag data for the 3.5 h interval were more repeatable (33%) and less variable (cv). Data were affected by T_a with a stronger effect on the externally attached transmitters, but the influence of wind speed was small for both methods. There was a significant positive relationship between transmitter‐ and PIT tag temperature during both days and nights. Both methods were equally suited to detect diel changes in peripheral T_b. However, transmitters offered longer detection distance and better temporal resolution. These qualities should be considered when deciding how to collect T_b data remotely. If properly deployed, both methods allow measurement of peripheral T_b over a wide range of natural systems and conditions in small, free‐ranging, birds.     

Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis

A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal.Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.     

Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis

A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal. Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.     

Feline panleukopenia viral infection in cats: Application of some molecular methods used for its diagnosis

Forty diseased cats and seven healthy control cats from different sex, ages and breeds had examined clinically to confirm presence or absence of clinical symptoms of Feline panleukopenia disease (FP). Several tools including ELISA, gene expression analysis (qRT-PCR), DNA fragmentation test and apoptosis assay were conducted to determine the Feline panleukopenia disease in cat tissues. Clinical symptoms in the form of depression, fever, anorexia, vomiting, diarrhea, dehydration, anaemia and leucopenia were recorded in the diseased cats while no clinical sings were observed in control healthy cats. ELISA results showed that all of diseased (n?=?40) cats were positive while control cats (n?=?7) were negative for FP viral antigen. After carrying out of ELISA assay, supportive treatment trials including fluid therapy, immunostimulant, antibiotics to overcome dehydration, restoring electrolytes imbalances, combating secondary bacterial infection were conducted but all diseased cats were died and control cats exposed to soft death. Gene expression analysis detected high levels of FP viral gene in several cat tissues in which ilium exhibited high viral expression levels compared with jejunum. Also, viral expression levels in jejunum were higher than in mesenteric lymph nodes. In addition, viral expression levels were not detected in tissues of control cats. The results of the DNA fragmentation assay observed that DNA extracted from different tissues of infected cats exhibited damaged DNA bands as compared with DNA of control cats. DNA fragmentation rates in infected tissues increased significantly (P?<?0.01), the highest rates were showed in ilium and jejunum tissue than in mesenteric lymph nodes. Determination of apoptosis in cat tissues showed that rate of apoptosis/necrosis increased significantly (P?<?0.05) in infected cats tissues in comparison to control cats. Moreover the highest apoptotic ratios of infected cats were observed in ilium and jejunum tissues compared with mesenteric lymph nodes.     

Evaluation of three serological tests for detection of anti-candidal antibodies in diagnosis of invasive candidiasis

Immunoblot detection of antibody against 47 KD cytoplasmic antigen ofCandida albicans was evaluated in diagnosis of invasive candidiasis and compared to whole cell agglutination and gel diffusion tests for detection of anticandidal antibody in 64 patients. The patients included 17 with culture proved candidemia, 34 with significant candiduria (more than 10,000 colony forming units per ml of urine) and 13 with nonsignificant candiduria. Antibody against 47 KD antigen was found to be the best indicator for diagnosis of invasive candidiasis even in patients with malignancy. The sensitivity of this procedure was 82.4%, specificity 86.7%, positive predictive value 77.8%, negative predictive value 89.7% and efficacy 85.1%. The gel diffusion procedure lacked in sensitivity whereas whole cell agglutination lacked in specificity. Detection of antibody against 47 KD antigen proved to be a valuable adjunct in the diagnosis of invasive candidiasis.     

A virus-type specific serological diagnosis of flavivirus infection using virus-like particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting flavivirus type.Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (ⅰ) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; (ⅱ)the neutralized VLPs are used to infect Vero cells; and (ⅲ) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT; importantly, it shortens the assay time from ＞10 days to ＜1 day, and can be performed in biosafety level-2 facility.