Blind Passage Research Articles

Evaluation of Cell Culture Methods for Detection of Largemouth Bass Virus

AbstractLargemouth bass virus (LMBV), a recently discovered iridovirus found in the eastern United States, is usually detected by isolation in cell culture. Although LMBV will replicate in several cell lines, optimal cell culture methods for the detection of this virus have not been determined. We tested inoculation method, adsorption time, incubation temperature, and various cell lines to determine the conditions that would provide the most sensitive cell culture assay for LMBV. The optimal inoculation procedure tested was to remove the culture medium from the culture well before the addition of the inoculum, and the optimal adsorption procedure tested was to allow the virus to adsorb for 40 min while the plates were on an orbital shaker. Following inoculation, incubation at 30°C resulted in a higher number of viral plaques than incubation at 25°C or 32°C. Four cell lines (bluegill fry (BF‐2), fathead minnow (FHM), epithelioma papulosum cyprini, and channel catfish ovary cells) inoculated with LMBV had similar susceptibility to infection. Similar percentages of LMBV‐positive samples were detected in BF‐2 and FHM cell cultures inoculated with homogenized organ samples from largemouth bass Micropterus salmoides; however, the use of two cell lines increased the number of infected samples discovered. A blind passage also increased the number of positive samples detected in cell culture. Subcultivation to confirm virus‐positive samples was useful for reducing false‐positive results.

Equine herpesvirus 2 (EHV-2) infection in thoroughbred horses in Argentina

BackgroundEquine herpesvirus 2 is a gamma-herpesvirus that infects horses worldwide. Although EHV-2 has been implicated in immunosuppression in foals, upper respiratory tract disease, conjunctivitis, general malaise and poor performance, its precise role as a pathogen remains uncertain. The purpose of the present study was to analyse the incidence of EHV-2 in an Argentinean horse population and correlate it with age and clinical status of the animals.ResultsA serological study on 153 thoroughbred racing horses confirmed the presence of EHV-2 in the Argentinean equine population. A virus neutralization test showed a total of 79.7 % animals were sero-positive for EHV-2. An increase in antibodies titre with age as well as infection at earlier ages were observed.EHV-2 was isolated from 2 out of 22 nasal swabs from horses showing respiratory symptoms. The virus grew slowly and showed characteristic cytopathic effect after several blind passages on RK13 cells. The identity of the isolates was confirmed by nested PCR and restriction enzyme assay (REA).ConclusionThis is the first report on the presence of EHV-2 in Argentina and adds new data to the virus distribution map. Though EHV-2 was isolated from foals showing respiratory symptoms, further studies are needed to unequivocally associate this virus with clinical symptoms.

Evaluation of Unpurified Cell Culture Supernatant as Template for the Polymerase Chain Reaction (PCR) with Largemouth Bass Virus

AbstractPolymerase chain reaction (PCR) has been used to identify largemouth bass virus (LMBV) in cell culture, and this method has included extraction of the sample DNA. To simplify this procedure, we centrifuged the cell culture fluid to remove cellular debris and then used the supernatant directly in the PCR without DNA extraction. For supernatants from cell cultures inoculated with 1:100 dilutions of homogenized swim bladder or pooled spleen and trunk kidney, only 70.0% of the LMBV‐positive samples (based on all available evidence) tested positive with this new PCR method. After a 1:500 dilution of these cell culture supernatants, 85% of the failed samples became PCR positive. In contrast, 97.2% of LMBV‐positive samples were PCR‐positive when undiluted supernatants from subcultivations (cells inoculated with supernatant from cell cultures showing cytopathic effect [CPE]) were used directly in the PCR. Unpurified cell culture fluid supernatant from blind passage samples (cells inoculated with supernatant from CPE‐negative cultures) was PCR‐positive for 93.8% of the samples that were eventually shown to be LMBV‐positive. The use of cell culture supernatant directly in the PCR reduced the cost and time required to identify LMBV isolated in cell culture. Extraction of DNA from cell cultures to obtain template for PCR identification of LMBV was required for only 2% of the LMBV‐positive samples.

Increased sensitivity of Trichomonas vaginalis isolation from vaginal secretions by subsequent blind passage of preliminary negative cultures

Trichomonas vaginalis is a vaginal protozoon causing the most prevalent nonviral sexually transmitted disease. Culture is considered as the most reliable and gold standard among conventional diagnostic methods in the detection of T. vaginalis. In the present study, the diagnostic effect of subsequent blind culturing of vaginal secretion samples in Diamond's complete medium at every 48 h was investigated in 93 women. Vaginal secretion samples were examined by wet mount and inoculated into Diamond's complete medium. All tubes were subcultured to a new culture tube blindly 5 times at 48-h interval and checked microscopically everyday for the presence of T. vaginalis trophozoites. Three of 93 women were found to be positive both by wet mount and standard culture methods. Five more positive results were detected with subsequent blind culturing among 90 negative results. This system appears to be a useful method for the detection of T. vaginalis.

Possibility for use of RT-PCR technique in establishing presence of bovine viral diarrhea virus in sperm of breeding bulls

The bovine viral diarrhea (BVD) virus is a significant health-economic pathogen in cattle which can be excreted and spread also through sperm of persistently or acutely infected bulls. Native sperm of 6 bulls, found to be negative to the BVD virus by isolating the virus and using the RT-PCR method, was experimentally infected with a tenfold dilution of the non-cytopathogen 22146 strain of the BVD virus with a titer of 105,5. This way, dilutions of the BVD virus from 10-1 to 10-6 (5 x 104 TCID/50 do 0,5 TCID/50 in 0.1 ml native sperm were obtained. From sperm infected in this way, the virus was reisolated on FTB cell culture in a microtiter plate with 96 pools in which each sample of the infected sperm was set up in three samples, and each of them was titrated to a dilution of 1:2 to 1:256. The presence of the BVD virus was proven using the technique of fluorescent antibodies in a second blind passage on FTB culture cells. For cell culture an extremely toxic effect of native sperm to a dilution of 1:64 was established. The BVD virus was reisolated from sperm in all three sperm samples with 5 x 104, 5 x 103 i 5 x 102 TCID/50, and it was not reisolated from sperm with 50, with 5, and with 0.5 TCID/50 BVD virus in 0.1 ml native sperm. At the same time, the presence of the BVD viral genome was proved using the RT-PCR method in the same samples of artificially infected native sperm of bulls. A positive re suit was established in native sperm with 5 x 104, 5 x 103, 5 x 102 and 50 TCID/50 BVD virus n 0.1 ml native sperm. The experiment proved that the RT-PCR method has advantages over the isolation of the BVD virus from samples of native sperm of bulls. These are: shortterm investigations (1 to 2 days) and greater sensitivity (10 times bigger than the isolation of the virus). The isolation of the virus takes at least 10 days, and its greater sensitivity is primarily a result of the cyrotoxic effect of native sperm of bulls on cell culture.

INTESTINAL PERFORATION: AN INFREQUENT COMPLICATION DURING INSERTION OF TENSION-FREE VAGINAL TAPE

The tension-free vaginal tape (TVT) surgical technique is a minimally invasive procedure that has demonstrated excellent long-term results for treating stress urinary incontinence (SUI) with a low complication rate.1 However, major intraoperative complications have been reported as a consequence of the blind passage of a trocar needle through the pelvic area.2, 3 We report a case of intestinal perforation during TVT placement, which represents the fifth case documented in the literature. CASE REPORT A 67-year-old female was hospitalized for elective surgical repair of SUI, a grade II cystocele and a grade II rectocele. The patient had a surgical history of cholecystectomy and vaginal hysterectomy. Rectocele and cystocele repair, as well as insertion of TVT, were performed with the patient under epidural anesthesia. Intraoperative cystoscopy showed no evidence of bladder perforation. The patient complained of right suprapubic and vaginal discomfort 12 hours postoperatively. Physical examination revealed a right suprapubic ecchymosis and a mild tenderness in the ecchymotic area without evidence of acute abdomen. On postoperative day 2 the patient had fever and tachycardia. Blood tests demonstrated increased blood urea nitrogen and serum creatinine levels of 43 mg/dl and 3.42 mg/dl, respectively (normal 7 to 20 and 0.8 to 1.0, respectively), and normal hematocrit and white blood count. Abdominopelvic ultrasound showed a right 2.4 cm paravesical collection. Effusion of intestinal material was seen draining through the right needle exit site. Abdominopelvic contrast enhanced computerized tomography (CT) revealed a right paravesical and suprapubic collection with air and intraperitoneal free air (fig. 1). Exploratory laparotomy demonstrated abundant small bowel adhered to the right paravesical space and a transfixing ileal perforation with the polypropylene mesh (fig. 2). Ileum resection and end-to-end ileal anastomosis were performed, as well as vaginal removal of the remaining polypropylene mesh. At 4-month followup the patient was continent and asymptomatic. DISCUSSION

Very virulent infectious bursal disease virus in Egypt: epidemiology, isolation and immunogenicity of classic vaccine.

Infectious bursal disease (IBD) is still a significant threat facing the Egyptian poultry industry. In this study, eight very virulent IBD viruses (vvIBDV), originating from acute IBD outbreaks recorded in lower and upper Egypt, were studied with respect to their ability to replicate in cell culture, antigenicity and immunogenicity of classic vaccine. Six continuous cell lines and one primary cell culture were tested for replication of the vvIBDVs. None of the vvIBDV isolates could be adapted to BGM-70, Vero, BHK, RK-13 or MDBK cell lines or chicken embryo fibroblast cells after six blind passages. Serological typing with three neutralizing monoclonal antibodies showed antigenic variation among vvIBDVs. The immunogenicity induced by graded doses of classic-intermediate vaccine in both IBD-resistant (Mandarah) and susceptible (Gimmizah) Egyptian chickens was investigated. The protection of the tested doses was evaluated by measurement of the serological response and resistance to vvIBDV challenge 10 days post vaccination. Similar antibody responses to the vaccine were generated over a wide (100-fold) dose range. It was concluded that single vaccination, by eye drops, with classic-intermediate vaccine (1 x) could protect chickens against clinical disease and mortality. However, the immune responses generated by 1 x, 10 x or 100 x vaccine doses did not protect against bursal damage following challenge. This finding points to the highly invasive nature of the prevailing vvIBDVs in Egypt.

An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype

The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs.

Experimental infection of California birds with western equine encephalomyelitis and St. Louis encephalitis viruses.

A total of 27 bird species from the San Joaquin and Coachella valleys of California were inoculated subcutaneously with sympatric strains of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses. Overall, 133 of 164 birds inoculated with WEE virus developed a viremia detected by plaque assay; significantly greater than 72 of 163 birds inoculated with SLE virus. Host competence was calculated as the average number of days that each avian species had a viremia > or = 2 log10 plaque-forming units per 0.1 ml, the threshold for infecting susceptible Culex tarsalis Coquillett, the primary vector of these viruses in California. Eleven of 20 species inoculated with WEE virus had a value > or = 1 and were considered to be competent hosts, whereas only six of 22 species inoculated with SLE virus had a value > or = 1. Overall, 133 of 164 birds inoculated with WEE virus and 105 of 163 inoculated with SLE virus produced antibody detectable by enzyme immunoassay and/or plaque reduction neutralization test. Six birds infected with WEE virus (one house finch, three mourning doves, one Brewer's sparrow, and one white-crowned sparrow) and nine birds infected with SLE virus (two house finches, three white-crowned sparrows, one song sparrow, two Western scrub-jays, and one orange crowned warbler) contained viral RNA detected by reverse transcription-polymerase chain reaction at necropsy > 6 wk postinoculation; infectious WEE and SLE viruses were only recovered from three mourning doves and an orange-crowned warbler, respectively, after blind passage in mosquito cells. Our study indicated that birds with elevated field antibody prevalence rates may not be the most competent hosts for encephalitis viruses and that relatively few birds developed chronic infections that could be important in virus persistence and dispersal.

ACCURACY OF INTRA‐ARTICULAR INJECTIONS FOR OSTEOARTHRITIS OF THE TRAPEZIOMETACARPAL JOINT

SUMMARYA study was conducted to determine the accuracy of intra‐articular injections for osteoarthritis (OA) of the trapeziometacarpal joint (TMCJ). Over a period of 12 months intra‐articular injections on 60 TMCJs (12 males and 48 females) were carried out. All the injections were performed in the operating theatre. The operator inserted the needle into the position clinically assessed to be the TMCJ and the position was assessed using biplanar fluoroscopy. In 42% of cases, the needle was incorrectly placed and its position had to be adjusted, using fluoroscopy, to ensure correct intra‐articular placement. Entry into an osteoarthritic TMCJ with a blind passage of the needle is not straightforward. A proper injection technique must be employed to enter the correct joint. Using an image intensifier greatly facilitates accurate needle placement. (Int J Clin Pract 2003; 57(4): 265–266)

Use of a gum elastic bougie (GEB) in a zone II penetrating neck trauma: a case report

A 54-year-old man presented with a deep zone II neck injury accompanied by profuse bleeding secondary to attempting suicide by slashing his anterior neck with a knife. Blind passage of the endotracheal tube (ETT) into the glottis through the open anterior neck was unsuccessful. In a second attempt a gum elastic bougie (GEB) was inserted directly through the vocal cord, and “tracheal clicking” and a “hold up” were appreciated. The ETT was then easily fed over the GEB and was successfully passed into the trachea. The patient underwent operative repair and tracheostomy, and he left the hospital 2 days later with his baseline mental status. The use and the benefits of the GEB are reviewed.

Toxoplasma gondii from liquid nitrogen for continuous cell culture:methods to maximise efficient retrieval

This study aims to increase the efficiency of continuous growth of Toxoplasma gondii in HeLa cells from tachyzoite stocks frozen in liquid nitrogen. Freezing and retrieval of tachyzoites for continuous cell culture requires more stringent protocols than those published for animal culture. The freezing and retrieval conditions are optimised so that a quality harvest (≥ 1 x 106 tachyzoites/mL, ≥ 90% viability) can be produced using T. gondii recovered from liquid nitrogen as fast and reliably as possible. Retrieval success rate increased from 36% to 100%. An improved freezing procedure using chilled reagents and freshly harvested parasites, and adoption of an effective recovery protocol with retrieval of 3 x 107 tachyzoites into 75cm2 flasks, change of maintenance media after six hours and subsequent blind passage all contributed to this success. The result is faster and more dependable production of T. gondii for diagnostic and experimental use.

The intubating laryngeal-mask airway may be an ideal device for airway control in the rural trauma patient

A review of the literature on advanced airway management indicates that the intubating laryngeal-mask airway (ILMA) may be an ideal device for airway control in the rural trauma patient. The ILMA is an advanced laryngeal-mask airway designed to allow oxygenation of the unconscious patient as well as blind tracheal intubation with an endotracheal tube. The ILMA is an easy-to-use airway with a high success rate of insertion, and requires little training. For the rural physician managing a difficult airway in a trauma patient, the ILMA has been found to be reliable and successful when other techniques fail, such as fiberoptic intubation and direct laryngoscopy. The ILMA has also been reported to cause less hemodynamic change and less injury to the teeth and lips than direct laryngoscopy. Further, the ILMA was found to be easier and faster to use with a higher success rate than either the combitube or endotracheal tube for unskilled healthcare providers. Limitations and complications of the ILMA may include aspiration, esophageal intubation, damage to the larynx or other tissues during blind passage of a tracheal tube, and edema of the epiglottis. (Am J Emerg Med 2003;21:80-85. Copyright 2003, Elsevier Science (USA). All rights reserved.)

Measles Virus Receptor SLAM (CD150)

Measles virus (MV), a member of the Morbillivirus genus in the Paramyxoviridae family, is an enveloped virus with a nonsegmented negative-strand RNA genome and infects humans and nonhuman primates (Griffin, 2001). Despite the availability of vaccines, MV remains a major cause of childhood mortality, claiming roughly one million lives a year worldwide. The transient immunosuppression that accompanies and follows measles renders the patients susceptible to secondary infections accounting for most of measles-related complications and deaths. MV also causes postinfectious encephalitis, and in rare instances, subacute sclerosing panencephalitis, a persistent infection in the central nervous system. This review is concerned with the identification of a new MV receptor and its implication for understanding the pathology and pathogenesis of MV infection. MV was first isolated in primary human kidney cells inoculated with the blood and throat washings of a child with measles (Enders and Peebles, 1954). This first isolate, the Edmonston strain, was subsequently adapted to chicken embryo fibroblasts and became the progenitor for currently used attenuated vaccines. The Edmonston strain also grows well in continuous cell lines and has become the most extensively studied MV strain in laboratories. Vero cells, an African green monkey kidney cell line, had been commonly used for MV isolation until a decade ago, but several blind passages were usually required before virus propagation and development of cytopathic effect (CPE). Kobune et al. (1990) reported that an Epstein–Barr virus (EBV)-transformed marmoset B cell line B95-8 and its adherent subline B95a were highly sensitive to MV. Furthermore, they showed that MV strains isolated in B95a cells, but not Vero cell-isolated

Endoscopic Treatment of Bladder Perforation After Tension-free Vaginal Tape Procedure

The tension-free vaginal tape procedure is a new technique for the treatment of female stress urinary incontinence.1 It is a simple, minimally invasive procedure performed with the patient under local or regional anesthesia and offers a 90% cure rate.2 However, despite its apparent simplicity, the procedure remains difficult to perform, and the blind passage of needles can be associated with significant complications. The most frequent complication associated with this procedure is bladder perforation. In a recent series of 404 cases a 6% rate of bladder perforation was noted.2 Usually, cystoscopic examination during surgery reveals the perforation and allows reorientation of the needle to a more lateral location. Leaving the bladder catheter indwelling for 48 hours permits the bladder to heal without consequences. However, in some cases, the perforation may be missed perioperatively, especially if the cystoscopic examination is not conducted with a 70 degree lens. In such cases solving the problem postoperatively may be difficult. An open operation to excise the tape has been described recently.3 Herein, a case of bladder perforation repaired by endoscopy is reported.

A Safe Method of Retrograde Passage of Fogarty Embolectomy Catheter Through Difficult Iliac Arteries

Retrograde instrumentation of the external and common iliac arteries may be difficult because of tortuosity, extensive exophytic disease or previous placement of a stent that may also be partially or totally occluded. In principle, the safest technique of intravascular manipulation of instruments is under angiographic imaging control using a catheter-/guidewire-directed technique. However, in practice, vascular surgeons may be faced with the need to negotiate a Fogarty embolectomy catheter up a difficult/stented iliac artery without the availability of either angiographic imaging control or catheter-/guidewire-directed technology. Blind and forceful passage of a Fogarty embolectomy catheter may result in dissection of a diseased native iliac artery or dislodgement of an iliac stent. We would like to report a method of pre-shaping the tip of a Fogarty Arterial Embolectomy Catheter, which allowed its safe passage up a stented iliac artery to successfully treat critical limb ischaemia due to thrombosed iliac stent.

Viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in ICAM-1-negative cells.

Major receptor group common cold virus HRV89 was adapted to grow in HEp-2 cells, which are permissive for minor group human rhinoviruses (HRVs) but which only marginally support growth of major-group viruses. After 32 blind passages in these cells, each alternating with boosts of the recovered virus in HeLa cells, HRV89 acquired the capacity to effectively replicate in HEp-2 cells, attaining virus titers comparable to those in HeLa cells although no cytopathic effect was observed. Several clones were isolated and shown to replicate in HeLa cells whose ICAM-1 was blocked with monoclonal antibody R6.5 and in COS-7 cells, which are devoid of ICAM-1. Blocking experiments with recombinant very-low-density lipoprotein receptor fragments and enzyme-linked immunosorbent assays indicated that the mutants bound a receptor different from that used by minor-group viruses. Determination of the genomic RNA sequence encoding the capsid protein region revealed no changes in amino acid residues at positions equivalent to those involved in the interaction of HRV14 or HRV16 with ICAM-1. One mutation was within the footprint of a very-low-density lipoprotein receptor fragment bound to minor-group virus HRV2. Since ICAM-1 not only functions as a vehicle for cell entry but has also a "catalytic" function in uncoating, the use of other receptors must have important consequences for the entry pathway and demonstrates the plasticity of these viruses.

Addition of Thoracoscopy to Nuss Pectus Excavatum Repair

Background and Purpose: Traditional repair of pectus excavatum (PE) has required a long and complex procedure with multiple rib resections and a large sternal scar. A minimally invasive procedure (the Nuss procedure) has recently been reported as an alternative method for PE repair. This operation involves blind passage of a clamp and rigid bar across the mediastinum, subjecting the patient to potential injury. The addition of thoracoscopy to this procedure is described in this report. Patients and Methods: During the 15 months from September 1998 to January 2000, we performed 32 PE repairs using the minimal access technique with thoracoscopic assistance. The thoracoscope is inserted into the right side of the chest for viewing during the repair. In three cases, bilateral thoracoscopy was performed. The patients ranged in age from 5 to 18 (average 10.7) years. Results: Thirty-one patients had one bar inserted under thoracoscopic guidance. One patient required two bars. The average operative time was 68 mi...

Ré-intubation difficile lors d’une trachéotomie de Fantoni résolue avec un masque laryngé

We report the failure of a reintubation under direct laryngoscopy, during a percutaneous tracheostomy by Fantoni method. After the accidental per-procedural extubation and in front of an unpredicted difficult conventional endotracheal intubation, a laryngeal mask was used as an airway intubator conduit for the blind passage of the setˈs ventilation tube. This ventilation tube is a 40 cm long, 4 mm internal diameter special catheter with low pressure cuff, included into the TLT™ kit. The end of the procedure was uneventful.

A prospective multicenter trial testing the SCOTI device for confirmation of endotracheal tube placement

We sought to characterize the Sonomatic Confirmation of Tracheal Intubation (SCOTI) device’s ability to confirm endotracheal tube location during real-time intubation in emergency and elective settings. Data were prospectively collected during a multicenter convenience-sample observational trial of emergency and elective intubation cases. In addition to tracheal and inadvertent esophageal intubations in emergency patients, intentional esophageal intubations were also performed to improve specificity calculations in consenting elective surgical patients. Data analysis included descriptive statistics as well as calculations of sensitivity, specificity, and 95% confidence intervals (CI). Data were obtained from 220 tracheal and 103 esophageal intubations, 137 (42%) performed in emergency patients. Fifteen tracheal intubations were incorrectly identified by SCOTI as esophageal and two esophageal intubations incorrectly as tracheal. Sensitivity and specificity were thus 93% (CI 90–97%) and 98% (CI 94–99%), respectively. The two false-positive cases were attributed to gaseous distension of the stomach and esophagus from prolonged bag-valve-mask ventilation. In addition to use in postprocedure tube placement confirmation, SCOTI aided the intubation procedure itself in 45 difficult emergency attempts (33%), 26 of which necessitated blind tube passage. We conclude that the SCOTI device has high sensitivity and specificity for tube placement confirmation during tracheal intubation attempts in both emergency and elective settings. It also facilitates tube placement itself during difficult intubations. As such it may be considered an adjunctive device to minimize the potentially fatal complication of esophageal intubation.