Urethroplasty performed with an autologous urothelium-vegetated collagen fleece to treat urethral stricture in the minipig model.

INTRODUCTION AND OBJECTIVE: Tissue-engineered materials in urethral reconstructive surgeries are a promising field for innovative therapy. Collagen matrices increase stability of cell-based implants and can promote viability and proliferation of urothelial cells. In this study, a collagen type I-based cell carrier (CCC) with stratified multi-layer autologous urothelium was used for urethroplasty after induction of urethral stricture in eight minipigs. MATERIALS AND METHODS: Minipigs underwent surgical procedures to induce urethral stricture by thermocoagulation. Simultaneously, bladder tissue was harvested. Urothelial cells were expanded, labeled with PKH26 and seeded onto CCC in high density. 3 weeks after strictures were induced and verified by urethrography, minipigs underwent urethroplasty using the seeded CCC. Two animals were euthanized after 1, 2, 4, and 24 weeks. Urethras were histologically examined for integration and survival of seeded CCC. In vivo phenotype of multi-layered urothelium matrix constructs was characterized via immunofluorescence staining with pancytokeratin, CK20, p63, E-cadherin and ZO-1. RESULTS: Seeded CCCs showed excellent stability and suturability after manipulation and application. Transplanted cells were detected using positive PKH26 fluorescence up to 6 months after labeling. Urothelium matrix implants integrated well into the host tissue without sign of inflammation. Animals showed no sign of rejection or stricture recurrence (urethrography) at any time during experimental period. Immunofluorescence analysis confirmed epithelial phenotype, junction formation and differentiation after 2 weeks. CONCLUSION: CCC can be suitable for urologic reconstructive surgeries and represents a promising option for clinical application. Longer follow-up results are required to exclude re-occurrence of stricture reformation.

Characterization of novel Mycobacterium tuberculosis pncA gene mutations in clinical isolates from the Ukraine.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.

Next-Generation Sequencing for Characterizing Drug Resistance-Conferring Mycobacterium tuberculosis Genes from Clinical Isolates in the Ukraine.

The Ukraine ranks among the top 20 countries with the highest number of multidrug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis cases in the world. However, little is known of the genetic diversity, i.e., resistance signatures, in clinical isolates from this region. We analyzed seven of most prevalent MDR/XDR antibiotic resistance-conferring genes from clinical isolates (n = 75) collected from geographically diverse Ukrainian oblasts and the southern Crimean peninsula. Genomic analysis revealed that 6 (8%) were sensitive, 3 (4%) were resistant to at least one antibiotic but were not MDR, 40 (53%) were MDR, and 26 (35%) were XDR. The majority of isolates (81%) were of the Beijing-like lineage. This is the first study to use next-generation sequencing (NGS) of clinical isolates from the Ukraine to characterize mutations in genes conferring M. tuberculosis drug resistance. Several isolates harbored drug resistance signatures that have not been observed in other countries with high-burden tuberculosis. Most notably, the absence of inhA gene promoter mutations, a diversity of mutation types in the rpoB resistance-determining region, and detection of heteroresistance provide a broader understanding of MDR/XDR from this area of the world.

Xpert(®) MTB/RIF detection of Mycobacterium tuberculosis from sputum collected in molecular transport medium.

BACKGROUND: The Xpert(®) MTB/RIF assay is widely used for Mycobacterium tuberculosis detection. However, specimen transport remains a challenge. PrimeStore Molecular Transport Medium(®) (PS-MTM) inactivates specimens and stabilizes DNA/RNA at ambient temperature for subsequent molecular detection. OBJECTIVE: To compare the detection of M. tuberculosis concentrations in PS-MTM using Xpert and real-time polymerase chain reaction (RT-PCR), and smear-positive sputum specimens collected using a flocked swab. METHODS: Dilutions of M. tuberculosis in PS-MTM and phosphate buffered saline (PBS) were analyzed using the Xpert assay and commercial RT-PCR. Smear-positive (1+ to 3+) sputum specimens (n = 17) were transferred by flocked swab into PS-MTM and PBS, and were compared to standard 1.0 ml sputum Xpert analysis. RESULTS: Using the Xpert assay, cycle threshold values from high M. tuberculosis concentrations in PS-MTM (>10(3) colony forming units [cfu]/ml) were increased compared to control. In contrast, M. tuberculosis samples containing <10(3) cfu/ml, i.e., low concentrations, suspended in PS-MTM resulted in detection down to 10 cfu/ml. Xpert detection efficiency in PS-MTM treated samples (63.2%) was improved compared to PBS controls (34.9%). Xpert detected M. tuberculosis in all sputum specimens collected by flocked swabs in PS-MTM, and correlated with routine Xpert detection. CONCLUSIONS: PS-MTM enhances M. tuberculosis detection at low concentrations of M. tuberculosis, and provides a simplified and efficient collection method for Xpert detection.

Molecular detection of Mycobacterium tuberculosis from sputum transported in PrimeStore(®) from rural settings.

SETTING: Mopani District, South Africa. OBJECTIVE: To explore remote, molecular detection of Mycobacterium tuberculosis from sputum transported using PrimeStore(®) Molecular Transport Medium (PS-MTM) compared to settings where microscopy or Xpert(®) MTB/RIF is used as the baseline test. DESIGN: Two sputum specimens were collected from patients with cough of ⩾ 2 weeks at clinics in rural South Africa. Shortly after expectoration and before processing using Xpert, microscopy and liquid culture, a flocked swab was swirled in each of these specimens and placed in PS-MTM. Swabs were stored and transported to the United States at ambient temperature for real-time PrimeMix(®) polymerase chain reaction (PM-PCR). RESULTS: Of 132 patients, 23 (17%) were positive on microscopy, 39 (30%) on Xpert and 44 (33%) by PS-MTM/PM-PCR. Concordance of PS-MTM/PM-PCR with positive microscopy and Xpert was respectively 96% and 85%. Of 107 microscopy-negative samples, 22 (21%) were positive using PS-MTM/PM-PCR, while 11/91 (12%) Xpert-negative samples were PS-MTM/PM-PCR-positive. PS-MTM/PM-PCR positivity was significantly higher than smear microscopy positivity (P < 0.001), but similar to Xpert (P = 0.33). CONCLUSION: PCR testing of specimens transported in PS-MTM would enhance TB diagnosis in settings where smear microscopy is the baseline diagnostic test, and could provide an alternative in settings where Xpert testing is not available.

A molecular transport medium for collection, inactivation, transport, and detection of Mycobacterium tuberculosis.

In many parts of the world, the diagnosis of tuberculosis (TB) has rapidly shifted to molecular detection and sequencing formats. The collection and transport of Mycobacterium tuberculosis specimens thus remains a challenging problem where TB is common and the infrastructure required for ensuring sample integrity is lacking. PrimeStore(®) Molecular Transport Medium (MTM) addresses this problem, rapidly inactivating/killing M. tuberculosis while preserving genomic DNA even at elevated temperatures for subsequent downstream molecular analysis.

Characterization of multi-drug resistant Mycobacterium tuberculosis from immigrants residing in the USA using Ion Torrent full-gene sequencing.

SUMMARY: Drug-resistant Mycobacterium tuberculosis bacterium (MTB) is spreading worldwide. Three drug-resistant isolates were detected in Burmese, Hmong, and Indian immigrants currently residing in Milwaukee, Wisconsin, USA. Ion Torrent full-gene sequencing and complete genetic analysis was performed within 5 days and compared to results from traditional drug sensitivity testing (DST). Genetic characterization of seven, full-length resistance-associated genes revealed two MDR and one highly resistant strain with important drug-resistant mutations that were confirmed by traditional DST. The rapid turnaround from sample-to-sequence underscores the public health value of Ion Torrent full-gene sequencing of MDR/XDR genes from epidemiologically significant clinical isolates.

Prostate carcinoma cell growth-inhibiting hydrogel supports axonal regeneration in vitro.

Prostate cancer is the most common malignant tumor in men. Radical prostatectomy, the most common surgical therapy, is typically accompanied by erectile dysfunction and incontinence due to severing of the axons of the plexus prostaticus. To date, no reconstructive therapy is available as the delicate network of severed nerve fibers preclude the transplantation of autologous nerves or synthetic tube implants. Here, we present an injectable hydrogel as a regenerative matrix that polymerizes in situ and thus, adapts to any given tissue topography. The two-component hydrogel was synthesized from a hydrolyzed collagen fraction and stabilized by enzymatic crosslinking with transglutaminase. Physical analysis employing osmolarity measurements and cryosectioning revealed an isotonic, microstructured network that polymerized within 2min and displayed pronounced adhesion to abdominal tissue. Cell culturing demonstrated the biocompatibility of the gel and a general permissiveness for various neuronal and non-neuronal cell types. No effect on cell adhesion, survival and proliferation of cells was observed. A chemotherapeutic drug was integrated into the hydrogel to reduce the risk of fibrosis and tumor relapse. Significantly, when the hydrogel was employed as a drug release depot in vitro, aversive fibroblast- and prostate carcinoma cell growth was inhibited, while axonal outgrowth from peripheral nervous system explants remained completely unaffected. Taken together, these results suggest that the gel's adequate viscoelastic properties and porous microstructure, combined with its tissue adhesion and neuritotrophic characteristics in the presence of a cell type-specific cytostatic, may constitute an appropriate hydrogel implant applicable to patients suffering from prostatectomy associated side effects.

A clinical specimen collection and transport medium for molecular diagnostic and genomic applications.

Pathogen detection and genetic characterization has dramatically changed in recent years. Clinical laboratories are transitioning from traditional culture and primer-specific sequencing to more robust and rapid nucleic acid testing such as real-time PCR and meta-genomic characterization, respectively. Specimen collection is the first step in any downstream molecular diagnostic procedure. PrimeStore Molecular Transport Medium (MTM) is an optimized blend of nucleic acid stabilizing reagents that includes a non-specific internal positive control that can be amplified using real-time RT-PCR for tracking the integrity of a specimen from the point of collection to detection. PrimeStore MTM is shown here to effectively kill pathogens, including highly pathogenic H5 influenza virus, inactivate nucleases and to protect and preserve released RNA at ambient temperature for up to 30 days for downstream real-time and traditional RT-PCR detection and genetic characterization. PrimeStore MTM is also compatible with a variety of commercial extraction kits. PrimeStore is suited for routine clinical specimens and has added utility for field collection in remote areas, triage centres, border crossings and during pandemics where cold-chain, transport, and dissemination of potentially infectious pathogens are a concern.

Molecular analysis of isolates from influenza B outbreaks in the U.S. and Nepal, 2005.

Currently circulating influenza B viruses can be divided into two antigenically and genetically distinct lineages referred to by their respective prototype strains, B/Yamagata/16/88 and B/Victoria/2/87, based on amino acid differences in the hemagglutinin surface glycoprotein. During May and July 2005, clinical specimens from two early season influenza B outbreaks in Arizona and southeastern Nepal were subjected to antigenic (hemagglutinin inhibition) and nucleotide sequence analysis of hemagglutinin (HA1), neuraminidase (NA), and NB genes. All isolates exhibited little reactivity with the B/Shanghai/361/2002 (B/Yamagata-like) vaccine strain and significantly reduced reactivity with the previous 2003/04 B/Hong Kong/330/2001 (B/Victoria-like) vaccine strain. The majority of isolates were antigenically similar to B/Hawaii/33/2004, a B/Victoria-like reference strain. Sequence analysis indicated that 33 of 34 isolates contained B/Victoria-like HA and B/Yamagata-like NA and NB proteins. Thus, these outbreak isolates are both antigenically and genetically distinct from the current Northern Hemisphere vaccine virus strain as well as the previous 2003-04 B/Hong Kong/330/2001 (B/Victoria lineage) vaccine virus strain but are genetically similar to B/Malaysia/2506/2004, the vaccine strain proposed for the coming seasons in the Northern and Southern Hemispheres. Since these influenza B outbreaks occurred in two very distant geographical locations, these viruses may continue to circulate during the 2006 season, underscoring the importance of rapid molecular monitoring of HA, NA and NB for drift and reassortment.

Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification.

Viral culture isolation has been widely accepted as the "gold standard" for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at -70 degrees C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

Development of real-time RT-PCR for the detection of avian influenza virus.

A real-time reverse transcriptase/polymerase chain reaction (RRT-PCR) assay was developed using hydrolysis probes for the detection of avian influenza virus (AIV) and the H5 and H7 subtypes. The AIV specific primers and probes were directed to regions of the AIV matrix gene that are conserved among most type A influenza viruses. The H5 and H7 primers and probes are directed to H5 and H7 hemagglutinin gene regions that are conserved among North American avian influenza viruses. The sensitivity and specificity of this RRT-PCR assay was compared to virus isolation (VI) in chicken embryos with 1550 clinical swab samples from 109 live-bird markets (LBMs) in New York and New Jersey. RRT-PCR detected influenza in samples from 61 of 65 (93.8%) of the LBMs that were the sources of VI positive samples. Of the 58 markets that were positive for H7 influenza by hemagglutination inhibition assay, RRT-PCR detected H7 influenza in 56 markets (96.5%). Too few H5 positive samples were obtained to validate the H5 RRT-PCR assay in this study. Although RRT-PCR was less sensitive than VI on an individual sample basis, this study demonstrated that the AIV and H7 RRT-PCR assays are good tools for the rapid screening of flocks and LBMs.

Assessment of the safety and efficacy of the monoclonal anti-tumor necrosis factor antibody-fragment, MAK 195F, in patients with sepsis and septic shock: a multicenter, randomized, placebo-controlled, dose-ranging study.

OBJECTIVE: To investigate the safety, biological effects, and efficacy of the anti-tumor necrosis factor (TNF) antibody fragment, MAK 195F, in a phase II trial in patient with severe sepsis. DESIGN: Prospective, randomized, open label, placebo-controlled, dose-ranging, multicenter, multinational clinical trial. SETTING: Sixteen academic medical centers' intensive care units in six European countries. PATIENTS: One hundred twenty-two patients with severe sepsis or septic shock who received standard supportive care and antimicrobial therapy. INTERVENTIONS: Patients received one of three different doses of the anti-TNF antibody (0.1 mg/kg, 0.3 mg/kg, or 1.0 mg/kg) or placebo; the antibody or placebo was given in nine doses at 8-hr intervals over 3 days. MEASUREMENTS AND MAIN RESULTS: There were no significant differences in mortality rates among the groups receiving various doses of the anti-TNF antibody or placebo, but patients with baseline serum interleukin (IL)-6 concentrations of > 1000 pg/mL appeared to benefit from MAK 195F in a dose-dependent fashion. Increased circulating IL-6 concentrations, but not TNF concentrations, were found to be important prognostic indicators for mortality for the patients in the placebo and the two lower dosage groups but not in the high dosage group (1 mg/kg). IL-6 concentrations decreased during the first 24 hrs of treatment in all three anti-TNF groups but not in the placebo group. MAK 195F was well tolerated by all patients. Human antimurine antibodies developed in 40% of the patients receiving the antibody. CONCLUSIONS: There was no increase in survival from sepsis for the patients receiving anti-TNF treatment in the overall study population. Retrospective stratification of patients by IL-6 concentrations suggests beneficial effects of the drug for patients with baseline circulating IL-6 concentrations of > 1000 pg/mL. This hypothesis requires validation in a larger, blinded, prospective study.

Effects of recombinant human tumor necrosis factor on rodent gliomas and normal brain.

In a study examining the possible therapeutic effects of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) on malignant gliomas without expression of tumor necrosis factor (TNF)-receptors, RG-2 glioma cells were tested in vitro as well as in a rat experimental glioma model. A growth inhibition assay revealed no inhibiting effect in vitro up to a concentration of 20 micrograms/ml rHuTNF-alpha. Receptor-binding studies showed that RG-2 cells did not present specific receptors for rHuTNF-alpha. The pharmacokinetics of rHuTNF-alpha after intravenous injection were studied with respect to serum, tissue, and brain tumor concentrations and showed increased glioma concentrations of (mean +/- standard error of the mean) 0.47 +/- 0.18 ng TNF/mg brain compared to 0.15 +/- 0.05 ng TNF/mg brain in the normal contralateral hemisphere. No therapeutic effect on solid RG-2 gliomas could be observed after stereotactic injection of 7.3 micrograms rHuTNF/10 microliter buffer solution into the tumor in 10 animals. Immunohistochemical studies after stereotactic injection of rHuTNF-alpha showed total disappearance of the substance after 24 hours without internalization into tumor cells. Stereotactic injection of 7.3 micrograms rHuTNF 10 microliters into normal brain resulted in marked inflammatory response around the injection track, including microvascular thrombosis. These results demonstrate that rHuTNF has neither direct nor indirect cytotoxic activity on RG-2 glioma cells. Furthermore, before clinical use of rHuTNF-alpha in malignant gliomas, the authors suggest that receptor studies be done in each patient. In receptor-positive patients undergoing treatment with rHuTNF-alpha, precautions should be taken to prevent local encephalitic reactions.

Low incidence of transplant-related complications in patients with chronic release of tumor necrosis factor-alpha before admission to bone marrow transplantation: a clinical correlate of cytokine desensitization?

To investigate the role of tumor necrosis factor-alpha (TNF-alpha) released by activated macrophages, sequential serum samples of 120 patients undergoing bone marrow transplantation (BMT) were analyzed by enzyme-linked immunosorbent assay. De novo increases in serum TNF-alpha levels were correlated with the development of acute endothelial complications as well as acute graft-versus-host disease. In addition, the analysis of time courses revealed a different capacity of TNF-alpha regulation at various phases of BMT. While patients with acute TNF-alpha release in the first 2 weeks of BMT had a significantly enhanced incidence of complications, a subgroup of 9 patients with chronic asymptomatic release of TNF-alpha before admission to BMT was observed. These patients were protected from complications in the course of the first 6 months of BMT. Our observations indicate the occurrence of desensitization for TNF-alpha, as it is also reported after repeated injections of TNF-alpha or endotoxin in experimental models.