Nanostructured Graphene Surfaces Promote Different Stages of Bone Cell Differentiation.

Nanostructured graphene films were used as platforms for the differentiation of Saos-2 cells into bone-like cells. The films were grown using the plasma-enhanced chemical vapor deposition method, which allowed the production of both vertically and horizontally aligned carbon nanowalls (CNWs). Modifications of the technique allowed control of the density of the CNWs and their orientation after the transfer process. The influence of two different topographies on cell attachment, proliferation, and differentiation was investigated. First, the transferred graphene surfaces were shown to be noncytotoxic and were able to support cell adhesion and growth for over 7 days. Second, early cell differentiation (identified by cellular alkaline phosphatase release) was found to be enhanced on the horizontally aligned CNW surfaces, whereas mineralization (identified by cellular calcium production), a later stage of bone cell differentiation, was stimulated by the presence of the vertical CNWs on the surfaces. These results show that the graphene coatings, grown using the presented method, are biocompatible. And their topographies have an impact on cell behavior, which can be useful in tissue engineering applications.

In Vitro Susceptibility of Mycobacterium tuberculosis to Amikacin, Kanamycin, and Capreomycin.

Amikacin, kanamycin, and capreomycin are among the most important second-line drugs for multidrug-resistant tuberculosis. Although amikacin and kanamycin are administered at the same dose and show the same pharmacokinetics, they have different WHO breakpoints, suggesting that the two drugs have different MICs. The aim of this study was to investigate possible differences in MICs between the aminoglycosides and capreomycin. Using the direct concentration method, a range of concentrations of amikacin, kanamycin, and capreomycin (0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, and 64.0 mg/liter) were tested against 57 clinical Mycobacterium tuberculosis strains. The 7H10 agar plates were examined for mycobacterial growth after 14 days. At 2 mg/liter, 48 strains (84%) were inhibited by amikacin and only 5 strains (9%) were inhibited by kanamycin (P < 0.05, Wilcoxon signed-rank test). The median MICs of amikacin, kanamycin, and capreomycin were 2, 4, and 8 mg/liter, respectively. No difference in amikacin, kanamycin, and capreomycin MIC distributions was observed between multidrug-resistant strains and fully susceptible strains. The results indicate that amikacin is more active than kanamycin and capreomycin against M. tuberculosis with the absolute concentration method. Determination of the impact of this difference on clinical outcomes in daily practice requires a prospective study, including pharmacokinetic and pharmacodynamic evaluations.

Molecular drug susceptibility testing in the Netherlands: performance of the MTBDRplus and MTBDRsl assays.

BACKGROUND: The performance of molecular drug susceptibility testing in countries with a low prevalence of drug resistance, such as the Netherlands, has not been adequately studied. OBJECTIVE: To evaluate the diagnostic accuracy of the GenoType(®) MTBDRplus and MTBDRsl assays to detect resistance to first- and second-line anti-tuberculosis drugs in the context of a nationwide screening programme in the Netherlands. RESULTS: The MTBDRplus assay had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100%, 99%, 80% and 100% for detecting rifampicin resistance. The sensitivity, specificity, PPV and NPV of either a katG or inhA mutation for detecting isoniazid resistance were 88%, 100%, 100% and 99%. The MTBDRsl assay had a sensitivity, specificity, PPV and NPV of 100%, 99%, 83%, and 100% for detecting moxifloxacin resistance; 62%, 71%, 58% and 74%, respectively, for detecting ethambutol resistance; 86%, 99%, 86% and 99% for detecting amikacin resistance; and 50%, 96%, 71% and 91% for detecting capreomycin resistance. CONCLUSION: The MTBDRplus and MTBDRsl assays may aid in decision making in tuberculosis treatment in low-level drug resistance settings and should preferably be used to exclude resistance.

Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm.

There is an urgent need for rapid and accurate diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis (MDR-TB). No diagnostic algorithm has been validated in this population. We hypothesized that pncA sequencing added to rpoB mutation analysis can accurately identify patients with pyrazinamide-resistant MDR-TB. We identified from the Dutch national database (2007-11) patients with a positive Mycobacterium tuberculosis culture containing a mutation in the rpoB gene. In these cases, we prospectively sequenced the pncA gene. Results from the rpoB and pncA mutation analysis (pncA added to rpoB) were compared with phenotypic susceptibility testing results to rifampicin, isoniazid and pyrazinamide (reference standard) using the Mycobacterial Growth Indicator Tube 960 system. We included 83 clinical M. tuberculosis isolates containing rpoB mutations in the primary analysis. Rifampicin resistance was seen in 72 isolates (87%), isoniazid resistance in 73 isolates (88%) and MDR-TB in 65 isolates (78%). Phenotypic reference testing identified pyrazinamide-resistant MDR-TB in 31 isolates (48%). Sensitivity of pncA sequencing added to rpoB mutation analysis for detecting pyrazinamide-resistant MDR-TB was 96.8%, the specificity was 94.2%, the positive predictive value was 90.9%, the negative predictive value was 98.0%, the positive likelihood was 16.8 and the negative likelihood was 0.03. In conclusion, pyrazinamide-resistant MDR-TB can be accurately detected using pncA sequencing added to rpoB mutation analysis. We propose to include pncA sequencing in every isolate with an rpoB mutation, allowing for stratification of MDR-TB treatment according to pyrazinamide susceptibility.

Susceptibility of clinical Mycobacterium tuberculosis isolates to a potentially less toxic derivate of linezolid, PNU-100480.

Susceptibility of clinical Mycobacterium tuberculosis isolates to PNU-100480 and linezolid was evaluated by the MGIT 960 system. The isolates had various susceptibilities to isoniazid (INH), rifampin, ethambutol, and streptomycin. The mean MIC for PNU-100480 was 3.2 times lower than that for linezolid. Therefore, PNU-100480 is a promising candidate to be developed further as an adjunct in the treatment of multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB).

Second-line drug resistance in multidrug-resistant tuberculosis cases of various origins in the Netherlands.

SETTING: The Netherlands. OBJECTIVE: To investigate the frequency of resistance to second-line drugs among multidrug-resistant tuberculosis (MDR-TB) cases and its correlation with patients' geographic origin. DESIGN: Retrospective laboratory database study of multidrug-resistant Mycobacterium tuberculosis complex strains isolated in the Netherlands between January 1993 and October 2007. RESULTS: We found 153 patients with MDR-TB, of whom 18 (12%) were native Dutch. Complete second-line drug susceptibility testing was performed for 131 MDR-TB patients. Resistance to second-line drugs was noted in primary samples of 28 (21%) MDR-TB patients. Resistance to a single second-line drug was most frequent (24/28 [86%]; 9 to prothionamide [PTH], 6 to para-aminosalicylic acid, 4 to amikacin [AMK], 4 to ciprofloxacin and 1 to cycloserine). Four MDR-TB patients had strains resistant to multiple second-line drugs; two were extensively drug-resistant M. bovis. In MDR-TB patients of European and Central Asian origin, resistance to second-line drugs was most frequent and involved the widest range of drugs. PTH resistance was frequent among African and American MDR-TB patients, while AMK resistance was frequent among South-East Asians. CONCLUSION: Resistance to second-line drugs is infrequent among MDR-TB patients in the Netherlands. Most second-line drug resistance is recorded among immigrants, with substantial differences in second-line drug resistance in MDR-TB patients originating from different geographical areas.

Divergent effect of bacillus Calmette-Guérin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: implications for primate models in tuberculosis vaccine research.

Despite the widespread use of bacillus Calmette-Guérin vaccination, Mycobacterium tuberculosis infection remains globally the leading cause of death from a single infectious disease. The complicated and often protracted dynamics of infection and disease make clinical trials to test new tuberculosis vaccines extremely complex. Preclinical selection of only the most promising candidates is therefore essential. Because macaque monkeys develop a disease very similar to humans, they have potential to provide important information in addition to small animal models. To assess the relative merits of rhesus and cynomolgus monkeys as screens for tuberculosis vaccines, we compared the efficacy of bacillus Calmette-Guérin vaccination and the course of infection in both species. Unvaccinated rhesus and cynomolgus monkeys both developed progressive disease with high levels of C-reactive protein, M. tuberculosis-specific IgG, and extensive pathology including cavitation and caseous necrosis. Bacillus Calmette-Guérin vaccination protected cynomolgus almost completely toward the development of pathology, reflected in a striking 2-log reduction in viable bacteria in the lungs compared with nonvaccinated animals. Rhesus, on the other hand, were not protected efficiently by the bacillus Calmette-Guérin. The vaccinated animals developed substantial pathology and had negligible reductions of colony-forming units in the lungs. Comparative studies in these closely related species are likely to provide insight into mechanisms involved in protection against tuberculosis.

Storage of mycobacterial strains.

The storage and maintenance of mycobacterial reference strains and clinical isolates are important parts of good laboratory practice in a mycobacterial laboratory. The storage of reference and control strains facilitates a reliable control on intra- and inter-test reproducibility (1,12). Furthermore, to study various aspects of the epidemiology of tuberculosis it is important to be able to study serial isolates of individual patients (13) or of patients associated with particular place or time factors (14).

Use of IS901 and IS1245 in RFLP typing of Mycobacterium avium complex: relatedness among serovar reference strains, human and animal isolates.

SETTING: Mycobacterium avium complex (MAC) includes major acquired immune deficiency syndrome (AIDS)-associated pathogens. Formerly, MAC serotyping was used for epidemiological purposes. Recently, restriction fragment length polymorphism (RFLP) typing has become available. OBJECTIVE: Examination of the usefulness of insertion sequence IS1245 in RFLP typing of MAC isolates and the association with IS901 RFLP. DESIGN: Ninety-four serovar reference strains were compared with 144 clinical and animal MAC isolates in RFLP typing. RESULTS: All but four strains containing M. avium-specific-rRNA possessed IS1245. Most human isolates showed polymorphic multiband IS1245 patterns, which were associated with serovars 4, 6 and 8. Sequential clinical isolates obtained at up to five years' distance displayed indistinguishable/closely related patterns. Eleven M. paratuberculosis isolates showed indistinguishable six-band patterns. All 29 MAC isolates from 23 bird species, 7/23 from mammals and 1/81 clinical isolates showed an IS1245 three-band pattern, associated with serovars 1, 2 and 3. All these IS1245 'bird' type strains showed closely related IS901 RFLPs. Only three IS1245 'non-bird' type strains contained IS901, but exhibited completely different RFLP patterns. CONCLUSION: IS1245-RFLP typing is useful for the classification of M. avium and epidemiology of most human isolates. The highly conserved IS901 and IS1245 RFLPs among 'bird' type isolates provide proof that these strains constitute a separate taxon within the MAC.

Antibiotic susceptibility of campylobacter isolates from sewage and poultry abattoir drain water.

In this study, the in vitro susceptibility of 209 campylobacter strains to the quinolones nalidixic acid, flumequine, ciprofloxacin, enrofloxacin, and to ampicillin, tetracycline and erythromycin was tested by the disk diffusion method. The strains were isolated from poultry abattoir effluent (DWA) and two sewage purification plants (SPA and SPB). Sewage purification plant SPA received mixed sewage, including that from a poultry abattoir, whereas SPB did not receive sewage from any meat-processing industry. The quinolone resistance of the DWA isolates ranged from 28% for enrofloxacin to 50% for nalidixic acid. The strains isolated from the sewage purification plants were more susceptible to the quinolones with a range of 11-18% quinolone resistance for SPB isolates to 17-33% quinolone resistance for SPA isolates. The susceptibility criteria as recommended by National Committee Clinical Laboratory Standards (USA) cannot readily be employed for campylobacter isolates. This investigation shows that the resistance of campylobacter bacteria is highest in the plant receiving sewage from a poultry slaughterhouse. Monitoring of antibiotic resistance of aquatic Campylobacter spp. is important, as surface waters are recognized as possible sources of infection.

Neonatal teratoma presenting as hygroma colli.

We describe a neonate with a large tumour involving cranial, cervical and upper mediastinal regions, which presented clinically as hygroma colli. Radiological and pathological investigations showed characteristics of a mature teratoma and prominent cystic components within the tumour. These findings suggest that during early fetal development primary lymphatic sacs were obstructed by a teratoma leading to hygromatous dilatations of lymphatic vessels or that the abnormal proliferation of lymphatic vessels (hygroma) was part of the teratoma, developing from mesoderm as one of the three germinal layers from which teratomas originate. A third possibility is that the cystic part of the tumour originated from plexus chorioideus tissue, containing CSF. The last possibility is most probable in this patient.

Carbon dioxide laser vaporization in early glottic carcinoma.

OBJECTIVE: Presently, widely employed treatment modalities for early glottic carcinoma include radiation therapy, surgical excision, and carbon dioxide laser excision. All these treatments have good oncological results, but poor or questionable functional results in terms of quality of voice and mucosal wave patterns as seen via a laryngostroboscope. We assessed the oncological and functional results of carbon dioxide laser vaporization of the diseased mucosa as a more conservative treatment alternative. DESIGN: Case series. SETTING: Department of Otolaryngology-Head and Neck Surgery in a tertiary care center. PATIENTS: Thirty-three patients with early glottic carcinoma (carcinoma in situ and Tla squamous cell carcinoma) who were referred to our center in the period from 1986 through 1990 were selected for carbon dioxide laser vaporization treatment on the criterion of a still recognizable mucosal wave pattern of the affected vocal fold via a laryngostroboscope. MAIN OUTCOME MEASURES: Local tumor control, voice quality, and a good appearance via a laryngostroboscope. RESULTS: Two patients were excluded from evaluation because a simultaneously diagnosed, incurable second primary tumor precluded frequent follow-up. Of the remaining 31 patients seen during a median follow-up period of 58 months, seven patients died of unrelated causes. A local recurrence of disease developed in two patients and was treated by radiation therapy. A recurrence of disease developed again in one of these patients but was successfully treated by total laryngectomy. Overall, local control of disease was achieved in all patients, with a 97% laryngeal preservation rate. Functional results in terms of voice quality were good in 97% of the patients, 75% of whom even retained a normal voice. Normal or near normal laryngostroboscopic appearance was achieved in 68% of all patients. CONCLUSION: Carbon dioxide laser vaporization is a good treatment alternative for early glottic carcinoma in terms of oncological as well as functional results.