Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production.

AIMS: The aim of this study was to develop and evaluate a real-time PCR technology for microbiological control methods to examine individualized cell therapeutics, an emerging class of pharmaceutical formulations. METHODS AND RESULTS: Oligonucleotide primers and hybridization probe for bacterial detection targeting the 16SrRNA gene were adapted based on Nadkarni et al. [Microbiology148 (2002) 257]. For detection of yeast and moulds, primers and probe were designed from conserved sequences of the 18SrRNA gene in this study. The real-time PCR assays were tested on genomic DNA of Escherichia coli and Candida albicans to assess efficiency and linear dynamic range. After successful establishment of robust real-time PCRs, applicability of the assays was evaluated by extracting microbial target DNA from cell-based preparations. Different commercial DNA extraction methods were compared identifying the MagNA Pure DNA Isolation Kit III as the method of choice. Sensitivity was examined for different strains and a detection limit of 102 -103 CFU per ml in a sample containing ~106 mammalian cells per ml was achieved. CONCLUSIONS: This study reports the successful establishment of two qualitative real-time PCR assays, enabling in general the broad-range detection of microbial contaminants in a cell-based sample matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Individualized cell therapeutics tend to have a short shelf life. Due to lengthy incubation periods, compendial testing according to current pharmacopoeial guidelines may not be applicable. We report a suitable alternative method upon which future microbiological quality control methods for such products could be based on. However, to implement valid rapid microbiological testing methods using real-time PCR technology, further challenges need to be addressed.

Treatment of testicular intraepithelial neoplasia (intratubular germ cell neoplasia unspecified) with local radiotherapy or with platinum-based chemotherapy: a survey of the German Testicular Cancer Study Group.

BACKGROUND: The treatment of testicular intraepithelial neoplasia (TIN), the progenitor of testicular germ cell tumours (GCTs), is based on little data. PATIENTS AND METHODS: Two hundred and twenty-eight GCT patients with contralateral TIN were retrospectively enrolled. Ten had surveillance, 122 radiotherapy to testis with 18-20 Gy, 30 cisplatin-based chemotherapy (two cycles), 51 chemotherapy (three cycles), and 15 carboplatin. The study end point was a malignant event (ME), defined as detection of TIN upon control biopsy or occurrence of a second GCT. The Secondary end point was hypogonadism during follow-up. RESULTS: Numbers, proportions of ME, and median event-free survival (EFS) times were: radiotherapy N = 3, 2.5%, 11.08 years; chemotherapy (two cycles) N = 15, 50%, 3.0 years; chemotherapy (three cycles) N = 12, 23.5%, 9.83 years; carboplatin N = 10, 66%, 0.9 years; surveillance N = 5, 50%, 7.08 years. EFS is significantly different among the groups. Hypogonadism rates were in radiotherapy patients 30.8%, chemotherapy (two cycles) 13%, chemotherapy (three cycles) 17.8%, carboplatin 40%, surveillance 40%. CONCLUSIONS: Local radiotherapy is highly efficacious in curing TIN. Chemotherapy is significantly less effective and the cure rates are dose-dependent. Though hypogonadism occurs in one-third of patients, radiotherapy with 20 Gy remains the standard management of TIN.

Methods to monitor distribution and metabolic activity of mesenchymal stem cells following in vivo injection into nucleotomized porcine intervertebral discs.

Intervertebral disc (IVD) degeneration involves a series of biochemical and morphological changes leading to loss of spinal stability and flexibility. Cell therapy is promising to reconstitute IVDs with new cells, however, sustained metabolic activity seems crucial for an active contribution to regeneration. The aim of the present study was to establish methods for separate follow up of persistence and activity of autologous porcine mesenchymal stem cells (pMSC) after implantation into IVDs of Goettingen minipigs in vivo in order to conclude about the potential of such an intervention strategy. For quantitative follow up, the transfer matrix was supplemented with Al(2)O(3) particles and pMSC which were retrovirally labeled with firefly luciferase (pMSC-Luc). Six mature Goettingen minipigs underwent matrix based cell transfer after partial nucleotomy of lumbar IVDs (n = 24). Day 0 and day 3 segments were analyzed for retained volume of Al(2)O(3) particles by micro-computed-tomography (muCT) and for cell activity by luciferase enzyme assessment. Three days after injection a reduction of Al(2)O(3) particles (P = 0.028) to about 9% and of pMSC-Luc activity to about 7% of initial values (P = 0.003) was detected, which suggests loss of 90% of the implant material under in vivo conditions without evidence for reduced pMSC-Luc metabolic activity (P = 0.887). In conclusion, separate follow up of implant material and cell activity was possible and unravels problems with in vivo implant persistence after annular puncture rather than quick loss of cell activity. Therefore, IVD-regeneration-strategies should increasingly focus on annulus reconstruction in order to reduce implant loss due to annular failure.

[Collagen hydroxyapatite (Healos) saturated with gentamicin or levofloxacin. In vitro antimicrobial effectiveness - a pilot study]. / Mit Gentamicin oder Levofloxacin gesättigter Kollagenhydroxyapatit (Healos). Antimikrobielle In-vitro-Wirksamkeit - eine Pilotstudie.

BACKGROUND: The functions of synthetic bone graft substitutes include not only structural support to provide bone healing and osseous ingrowth but also the ability to serve as a local antibiotic delivery system to prevent or treat infections of the spine. MATERIAL AND METHODS: The impregnation and antibiotic efficiency of gentamicin and levofloxacin with Healos was investigated in vitro and compared with Healos without an antibiotic additive. These antibiotic-loaded bone graft substitutes were examined without dilution and with 10-fold and 100-fold dilution for activity against spondylodiscitis-causing bacteria on different agar plates using an agar diffusion method. RESULTS: All hydroxyapatite (HA)/collagen-saturated diluted antibiotics showed elliptical inhibition zones on the corresponding agar plates. For both antibiotics, there was a linear correlation between dilution and area of the inhibition zone. CONCLUSION: The analysis showed that the antimicrobial activity of HA/collagen-saturated antibiotics corresponded to the antimicrobial dilutions. These results should be further analyzed using in vivo studies to determine the remaining antibiotic efficiency after implantation of bone graft substitutes.

Cottonmouth snake bites reported to the ToxIC North American snakebite registry 2013-2017.

Introduction: The majority of venomous snake exposures in the United States are due to snakes from the subfamily Crotalinae (pit vipers). There are three types of US pit vipers: rattlesnakes (Crotalus and Sisturus spp.) copperheads (Agkistrodon contortrix), and cottonmouths (Agkistrodon piscivorus) also known as water moccasins. Cottonmouth bites are reported less frequently than other pit viper envenomations, and data on cottonmouth envenomation are limited. Our objective was to describe the epidemiology, clinical manifestations, and management of cottonmouth envenomations using prospective data reported to the Toxicology Investigators Consortium's (ToxIC) North American Snakebite Registry (NASBR)Methods: Cottonmouth envenomation cases reported to NASBR for the period from January 1, 2013, through December 31, 2017 were reviewed. Variables collected included patient demographics, bite location, clinical manifestations, and management.Results: Thirty-one cottonmouth envenomations were reported. Most bites occurred in children aged 7-12 (39%). Most bites involved the lower extremity (72%). Intentional interaction with the snake occurred in three cases (10%). Swelling was the most reported clinical effect and occurred in all patients. Gastrointestinal symptoms were reported in 19% of patients, and 19% developed coagulopathy. Antivenom treatment was used in 84% of patients. Nineteen patients (61%) required hospital stays of >24 hours.Discussion: Our study represents the first systematic prospective data collection on cottonmouth bites. Our data demonstrate that cottonmouth envenomations cause primarily local effects and, occasionally, systemic toxicity. Our study also demonstrates that antivenom is often indicated for these envenomations per published guidelines and recommendations.Conclusions: Cottonmouth envenomations are relatively infrequent. However, they can cause significant local and systemic toxicity. Most cottonmouth envenomations in this series were treated with antivenom and were hospitalized beyond 24 hours.

[The German diagnosis-related groups in urology: reproducibility and quality of coding]. / G-DRG-System für die Urologie : Reproduzierbarkeit und Kodierqualität.

BACKGROUND: The German diagnosis-related group (G-DRG) system is based on the belief that there is only one specific coding for each case. The aim of this study was to compare coding results of identical cases coded by different coding specialists. MATERIAL AND METHODS: Charts of six anonymous cases -- except final letter and coding -- were sent to 20 German departments of urology. They were asked to let their coding specialists do a DRG coding of these cases. The response rate was 90%. RESULTS: Each case was coded in a different way by each coding specialist. The DRG refunding varied by 6-23%. The coding differences were caused by different interpretations of definitions in the DRG system and also by inaccurate chart analysis. CONCLUSION: The present DRG system allows a wide range of interpretation, leading to aggravation of the ongoing disputes between hospitals and insurance companies.

Two cases of intoxication with new synthetic opioid, U-47700.

BACKGROUND: Novel substances often referred to as "designer drugs" have emerged as drugs of abuse, and recognition of these is difficult as routine blood and urine screening tests do not detect these agents. U-47700 is a synthetic selective µ-opioid agonist that can be bought online for as little as $40 per gram. We report two patients presenting after insufflation of U-47700, with subsequent confirmation of this substance in urine samples. CASE DETAILS: A 26-year-old man and 24-year-old woman insufflated a substance they believed to be "synthetic cocaine." The man was found down with cyanosis and agonal respirations. He was intubated and taken to hospital where he recovered well with supportive care. The woman presented with anxiety, tremors and drowsiness and was admitted for observation. Urine samples from both patients were analyzed using GC/MS/MS and LC/QToF, and U-47700 was isolated in both cases. No other opioids were detected. DISCUSSION: These cases are concerning because U-47700 is a relatively new agent that is easy to obtain over the internet and has the potential to cause significant morbidity and mortality.

Changing diagnostic and therapeutic concepts in high-flow priapism.

High-flow priapism (HFP) is defined as pathological increased arterial influx into the cavernosal bodies. Since 1960, 202 cases have been published in the literature. This study evaluates the effect of the changing diagnostic and therapeutic concepts. The data of 202 cases of HFP was evaluated regarding diagnostic and therapeutic procedures and long-term results. Success was defined as restored erectile function without recurrent priapism. The major etiology of HFP is trauma, especially in children or young adults; in older men, HFP is a rare event mainly caused by malignoma. Cavernosal blood-gas analysis, color-Doppler ultrasound and angiography were the most effective diagnostic tools to distinguish high- from low-flow priapism. The success rate was 20% for shunt operations and 89% for arterial embolization. In conclusion, embolization was effective in the majority of cases of traumatic HFP, while shunt surgery remained disappointing. For HFP caused by inherited diseases and malignoma conservative therapy is mandatory.

A human arterial organ culture model of postangioplasty restenosis: results up to 56 days after ballooning.

BACKGROUND: Restenosis is a reparative process that is activated in response to injury induced by angioplasty. Despite numerous experimental models of restenosis the number of human arterial organ culture systems is very limited and long-term experiences do not exist. METHODS AND RESULTS: During routine nephrectomies parts of the renal arteries of 88 patients were extracted, 47 were suitable for organ culture preparations. Sections were made at 3 mm intervals perpendicular to the vessel wall axis. The arterial segments were treated with 3 mm standard balloon-catheters (Medtronic 14K2030E) for 60 s with 3, 6, 9, and 12 bar. After angioplasty, the organ segments were cultured in a mixture of Waymouth's MB 752/1 and Ham F-12, supplemented with 15% fetal calf serum. After 0, 4, 14, 21, 28, and 56 days the organ cultures were fixed in 4% para-formaldehyde and embedded in paraffin. After staining with a modified elastica-van Gieson technique the intimal wall thickening was analyzed with a computerized morphometric system. For the identification of smooth muscle cells (SMC) a monoclonal antibody against smooth muscle alpha-actin was used. Endothelial cells were identified using an anti-human von Willebrand factor. To determine the number of cells undergoing DNA synthesis, bromodeoxyuridine (BrdU), a thymidine analogue, was added to the culture media 18 h prior to fixation. BrdU was detected with a monoclonal antibody, as secondary antibody a biotinylated horse-anti-mouse antibody was used. After 14, 21, and 28 days in culture BrdU-positive cells were detected in the neointima of the organ cultures, indicating mitotic activity in this area. After 28 and 56 days in culture a clear increase of neointimal thickening was found in the morphometric analysis. By positive reaction with antibodies against smooth muscle alpha-actin these cells were partly identified as SMC. CONCLUSIONS: The organ culture model offers opportunities for in vitro investigations of postangioplasty restenosis. The data emphasize the importance of a relatively late proliferative response of SMC in the human arterial organ culture model.

Serum half-life time determination of free and total prostate-specific antigen following radical prostatectomy--a critical assessment.

OBJECTIVES: All studies investigating the elimination kinetics of serum total (tPSA) and free (fPSA) prostate-specific antigen (PSA) were carried out in men undergoing radical prostatectomy. Radical prostatectomy itself could, however, have a major influence on the serum concentration of these tumor markers (e.g., perioperative fluid shift or blood loss). The purpose of our study was to determine the half-life time of fPSA and tPSA with special regard to the influence of the radical prostatectomy on the serum concentration of these tumor markers. METHODS: Eleven men (mean age 63.2+/-7.2 years) with organ-confined prostate cancer who underwent radical prostatectomy were investigated (final pathologic Stage pT2pN0 or lower). Serum samples were obtained preoperatively and 0.25, 0.5, 1, 2, 4, 8, 12, 16, 24, 48, 72, 120, 168, and 240 hours after removal of the prostate. fPSA and tPSA and albumin and total protein serum concentrations were determined in all samples. RESULTS: During the first 120 minutes after removal of the prostate, albumin and total protein serum concentrations continuously declined, with a half-life time of -104.5+/-28 minutes and -129.7+/-32 minutes, respectively. Serum decline of fPSA and tPSA followed a biphasic kinetic. During the initial alpha-phase, fPSA and tPSA serum concentrations decreased, with a half-life time of -69+/-10.3 minutes and -87.3+/-18.1 minutes, respectively. During the terminal beta-phase, the half-life time of fPSA and tPSA was -1152.2 minutes (0.8 days) and -3916.1 minutes (2.7 days), respectively. Between the alpha-phase half-life time of fPSA or tPSA and the half-life time of the total protein or albumin concentration decline, significant correlations were found. CONCLUSIONS: These correlations indicate that the rapid decline of fPSA and tPSA directly after removal of the prostate (alpha-phase half-life time) is caused by the radical prostatectomy itself. The half-life time of the beta-phase reflects the biologic clearance of PSA. Therefore, the half-life time determination of PSA after radical prostatectomy is of limited value if the influence of the operation itself on the serum PSA concentration is not taken into account.

Rheological changes in the blood of patients with chronic arterial occlusive disease after the administration of vasoactive drugs.

Patients with chronic arterial occlusive disease were found to have higher blood and plasma viscosity levels than healthy controls. Measurements were taken at high and low shear rates. Blood and plasma viscosity in these patients was significantly reduced following parental administration of pentoxifyline, and this effect was particularly pronounced at low shear rates. The changes recorded after administriation of bencyclane did not follow a consistent pattern. There was no significant correlation between the rheological changes measured and changes in plasma protein fractions.

The ileal neobladder. Operative technique and results.

Bladder substitution by continent bowel reservoirs has been one of the real advancements in modern urology. Short-term experience with and patients' acceptance of the ileal neobladder have been excellent. As do others, we consider this technique to date the procedure of choice in male patients requiring radical cystectomy.

Biochemical relapse of prostate cancer. Evidence after radical surgery.

BACKGROUND: The aim of this study was to investigate, whether ultrasensitive PSA assays can help to find a lower cut-off value for biochemical recurrence of prostate cancer after resection. MATERIALS AND METHODS: This was a retrospective analysis of the patient files of 113 men with prostate cancer who underwent surgery. The mean follow-up time was 26 months. PSA measurements were performed with the ultra sensitive Immulite assay (DPC, USA), with an analytical sensitivity of 0.001 ng PSA/ml. Patients with baseline PSA levels > 0.1 ng/ml after surgery were excluded. Different cut-off levels for PSA were applied on the values and PSA doubling-times calculated. RESULTS: Maximum PSA 0.01 ng/ml, 0.05 ng/ml, 0.1 ng/ml, 0.2 ng/ml and > 0.4 ng/ml were reached by 87%, 61%, 50%, 42% and 20% of patients, respectively. From a cut-off point of 0.1 ng/ml the chance of further progression to PSA levels of 0.2 ng/ml and 0.4 ng/ml was 75% and 40%, respectively. The PSA doubling time was 2.1 months in patients with PSA values > 0.4, 15 months for patients below 0.4 ng/ml and 22.4 months for patients whose PSA never exceeded 0.1 ng/ml. CONCLUSION: PSA cut-off values below 0.4 ng/ml lack sufficient clinical significance. However, patients with PSA values > 0.1 ng/ml should be monitored thoroughly and at shorter intervals, as further increase, i.e. tumour progression, is very likely to occur.

Level I trauma certification and emergency medicine resident major trauma experience.

OBJECTIVE: American College of Surgeons (ACS) and Residency Review Committee for Emergency Medicine (RRC-EM) guidelines conflict regarding the role of emergency physicians in directing major trauma resuscitations. This article describes the impact of ACS level I trauma certification on emergency medicine (EM) resident trauma experience. METHODS: A written survey and a follow-up letter were sent to all 101 EM program directors as of August 16, 1994. The survey addressed demographics and trauma experience at hospitals designated by the RRC-EM as primary training sites. RESULTS: There were 95 (94%) survey respondents. Estimates of the percentage of trauma resuscitations directed by EM residents were significantly lower at level I centers (52% +/- 27%, 95% CI 45-59%) than they were at non-level I centers (70% +/- 30%, 95% CI 58-82%) (p < 0.01). There was no significant difference in trauma census between level I and non-level I centers. Of 14 respondents who said they were cited by the RRC-EM for inadequate trauma experience, ten (71%) were in ACS level I trauma centers (p = 1.0). Twelve of the 14 respondents cited for inadequate trauma experience were in either the Northeast or the Midwest. CONCLUSIONS: EM residents direct a smaller percentage of major trauma resuscitations at ACS level I hospitals than they do at non-level I facilities. This finding is not offset by an increased trauma census at level I facilities and may be more pronounced in the Northeast and the Midwest.

Interobserver variability among faculty in evaluations of residents' clinical skills.

OBJECTIVE: To describe interobserver variability among emergency medicine (EM) faculty when using global assessment (GA) rating scales and performance-based criterion (PBC) checklists to evaluate EM residents' clinical skills during standardized patient (SP) encounters. METHODS: Six EM residents were videotaped during encounters with SPs and subsequently evaluated by 38 EM faculty at four EM residency sites. There were two encounters in which a single SP presented with headache, two in which a second SP presented with chest pain, and two in which a third SP presented with abdominal pain, resulting in two parallel sets of three. Faculty used GA rating scales to evaluate history taking, physical examination, and interpersonal skills for the initial set of three cases. Each encounter in the second set was evaluated with complaint-specific PBC checklists developed by SAEM's National Consensus Group on Clinical Skills Task Force. RESULTS: Standard deviations, computed for each score distribution, were generally similar across evaluation methods. None of the distributions deviated significantly from that of a Gaussian distribution, as indicated by the Kolmogorov-Smirnov goodness-of-fit test. On PBC checklists, 80% agreement among faculty observers was found for 74% of chest pain, 45% of headache, and 30% of abdominal pain items. CONCLUSIONS: When EM faculty evaluate clinical performance of EM residents during videotaped SP encounters, interobserver variabilities are similar, whether a PBC checklist or a GA rating scale is used.

Reproducibility of a single-sample method for 99Tcm-MAG3 clearance under clinical conditions.

The aim of this study was to assess the intra-individual reproducibility of 99Tcm-mercaptoacetyltriglycine (99Tcm-MAG3) clearance in routine clinical practice in various circumstances using a single-sample method. Three groups of 30 patients each were enrolled in the study. Investigations were repeated on the same day with two different levels of activity (group A), within 1 week (group B), or after 1 year (group C). Blood samples were taken after 20 and 25 min and clearance values were averaged to minimize incidental errors after calculation according to the formula proposed by Bubeck et al. For clearance estimation on the same day, time-dependent background correction for residual activity was carried out. The mean difference was -2.0%, +6.0% and -1.5% (S.D. = 6.3%, 15.7% and 11.7%) for groups A, B and C respectively, taking into account values > 100 ml min-1 only. We demonstrated that 99Tcm-MAG3 clearance is highly reproducible under the same conditions. However, a repeat clearance measurement on a separate day revealed large differences. Therefore, in follow-up studies for evaluation of changing kidney function (e.g. surgery or chemotherapy), differences should exceed 30% (i.e. 2 S.D. of group B) to be statistically significant.

Pharmacology of low molecular weight heparins.

Although intravenous heparin has been the treatment of choice for acute VTE disease, LMWHs are gaining wider recognition and support as not only a new option but also as the standard of care. Each LMWH is viewed as a unique drug by regulatory agencies because of their differing physical and pharmacokinetic attributes. LMWHs have high absorption, high bioavailability, and long half-lives enabling once- or twice-daily dosing with predictable dose-response relationships. These factors enable the LMWHs to be used without laboratory monitoring and at home for acute DVT management. Studies continue to show that LMWH preparations are as at least as effective as heparin in a variety of settings, including VTE disease prophylaxis, management of acute VTE disease, unstable angina, and NSTEMI. They are at least as safe as heparin relative to hemorrhagic complications. Heparin-induced thrombocytopenia is less of a problem with LMWHs. Use of LMWHs has resulted in cost benefits in the treatment of acute DVT, unstable angina, and NSTEMI as well as in prophylaxis against venous thromboembolism. Emergency physicians, because of their unique position at the forefront of acute care, will soon regularly use LMWHs.

[Early detection of a contralateral second carcinoma in patients with testicular tumors caused by testicular carcinoma in situ]. / Früherkennung des kontralateralen Zweitkarzinoms bei Hodentumorpatienten durch das Carcinoma in situ testis.

Carcinoma in situ (CIS) of the testis is considered to be a precursor of germ cell cancer. Diagnosis is made by the conventional biopsy technique. Only for patients at risk is a screening biopsy justifiable. This group includes patients with testicular cancer in whom the incidence of contralateral second germ cell tumors is increased. In a prospective study we found three cases of testicular CIS in biopsies from the contralateral testes of 61 such patients. All cases with a diagnosis of CIS presented with testicular atrophy (volume less than 12 ml), associated with necrozoospermia in one patient and with azoospermia in two patients. Treatment consisted in local irradiation (20 Gy) of the remaining testis to preserve Leydig cell function. In control biopsies no evidence of CIS or germ cells was found. More than 3 months after therapy, plasma testosterone levels were normal and LH and FSH levels were increased. None of the patients with negative biopsy (n = 49) who were followed up was found to have a second cancer of the contralateral testis. The average observation time so far is 17.2 months.

[Intra- and postoperative morbidity of radical prostatectomy]. / Intra- und postoperative Morbidität der radikalen Prostatektomie.

Operation-related morbidity was analyzed in 147 patients, who had undergone radical prostatectomy during the last 3 years. Six major complications (4.1%) had occurred requiring surgical revision. One single postoperative death (0.7%) was caused by a pulmonary embolus. In 51 patients (34.7%) minor complications were noted. Urinary incontinence was severe in 4 patients (3.9%), which was treated by an artificial sphincter in one man during the follow-up period. Twelve patients (11.8%) complained of stress incontinence, grade I-II. Postoperative determinations of serum PSA were below the detectable range in 81% of the patients, including those with a locally advanced tumor stage and adjuvant antiandrogen therapy. Based on our data, it is concluded that radical prostatectomy is today a safe and standardized operative procedure.

[PSA volume quotient: an additional parameter in diagnosis of locally confined prostate cancer]. / PSA-Volumenquotient: Ein zusätzlicher Parameter in der Diagnostik des lokal begrenzten Prostatakarzinoms.

The sensitivity and specificity of prostate-specific antigen density (PSAD), a quotient of serum PSA and prostate volume, in the detection of localized prostate cancer was analysed in a prospective study. A total of 235 patients were examined, 145 without prostate cancer and 90 patients before radical prostatectomy for localized prostate cancer. PSAD was determined by dividing the serum level of PSA by the volume of the entire prostate (estimated by transrectal ultrasound) and multiplying by 100. Using a PSAD of 15, the specificity achieved in our collective was the same as with an absolute PSA value of 4 ng/ml (88.9-90%). On the other hand, with the PSAD of 15 we also found the same sensitivity as with an ab-solute PSA of 10 ng/ml (75.2-76.5%).