Three-dimensional characterization of in vivo intervertebral disc degeneration using EPIC-µCT.

OBJECTIVE: Small animal models are commonly employed to study progression of and potential treatment techniques for degenerative disc disease (DDD), but assessment using conventional imaging techniques is challenging due to resolution. The objective of this study was to employ equilibrium partitioning of an ionic contrast agent micro computed tomography (EPIC - µCT) to map three-dimensional (3D) degenerative changes in the rabbit intervertebral disc (IVD). MATERIALS AND METHODS: In vivo degeneration was induced surgically in 12 New Zealand White rabbits via percutaneous annular puncture and percutaneous nucleotomy. IVDs were harvested after 3 and 6 weeks. EPIC-µCT imaging was performed on fresh, IVDs before and after formalin fixation, and 3D IVD volumes were segmented. IVDs were histologically stained with Safranin-O/Fast-Green and Hematoxylin & Eosin (H&E). EPIC-µCT attenuation and 3D morphological measurements were assessed in healthy and degenerate IVDs and compared to qualitative grading and disc height measurement from histology. RESULTS: EPIC-µCT caused pronounced contrast enhancement of the IVD. Annular puncture and nucleotomy produced mild and severe degenerative changes, respectively. IVD attenuation following contrast enhancement increased significantly in nucleotomized discs at 3 and 6 weeks. IVD attenuation correlated significantly with histologic score and disc height measurements. Disc height decreased most extensively in the posterior and lateral aspects of the IVD. 3D morphological measurements correlated strongly to IVD attenuation and were more sensitive to degenerative changes than histologic measurements. Formalin fixation reduced the attenuation of IVDs by ∼10%. CONCLUSION: EPIC-µCT is sensitive to in vivo DDD induced by nucleotomy and provides a high resolution 3D method for mapping degenerative changes in rabbit IVDs.

Fitness cost associated with resistance to fluoroquinolones is diverse across clones of Klebsiella pneumoniae and may select for CTX-M-15 type extended-spectrum ß-lactamase.

Lowered fitness cost associated with resistance to fluoroquinolones was recently demonstrated to influence the clonal dynamics of methicillin-resistant Staphylococcus aureus (MRSA) in the health care setting. We investigated whether or not a similar mechanism impacts Klebsiella pneumoniae. The fitness of K. pneumoniae isolates from major international hospital clones (ST11, ST15, ST147) already showing high-level resistance to fluoroquinolones and of strains from three minor clones (ST25, ST274, ST1028) in which fluoroquinolone resistance was induced in vitro was tested in a propagation assay. Strains from major clones showed significantly less fitness cost than three of four fluoroquinolone-resistant derivatives of minor clone isolates. In addition, plasmids with CTX-M-15 type extended-spectrum ß-lactamase (ESBL) genes were all retained in both major and minor clone isolates, irrespective of the strains' level of fluoroquinolone resistance, while each plasmid harboring SHV-type ESBLs had been lost during the induction of resistance. Major clone K. pneumoniae strains harbored more amino acid substitutions in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes than minor clone isolates. The presence of an active efflux system could be demonstrated in all fluoroquinolone-resistant derivatives of originally SHV-producing minor clone isolates but not in any CTX-M-15-producing strain. Further investigations are needed to expand and confirm our findings on a larger sample. In addition, a long-term observation of our ciprofloxacin-resistant minor clone isolates is required in order to elucidate whether or not they are capable of restoring their fitness while concomitantly retaining high minimum inhibitory concentration (MIC) values.

Significance of methicillin-teicoplanin resistant Staphylococcus haemolyticus in bloodstream infections in patients of the Semmelweis University hospitals in Hungary.

The purpose of this study was to quantify the impact of Staphylococcus haemolyticus in the epidemiology of the blood stream infection (BSI) and to characterize the rates and quantitative levels of resistance to antistaphylococcal drugs. During an eight-year period, 2967 BSIs of the patients hospitalized in different clinical departments of the Semmelweis University, Budapest, Hungary were analyzed. One hundred eighty-four were caused by S. haemolyticus, amounting to 6% of all infections. The antibacterial resistance of S. haemolyticus isolates was investigated by the broth microdilution method, vancomycin agar screen, population analysis profile and PCR for mecA, vanA and vanB genes detection. Epidemiological investigation was processed by determining phenotypic antibiotic resistance patterns and PFGE profiles. Extremely high MIC levels of resistance were obtained to oxacillin, erythromycin, clindamycin, gentamicin and ciprofloxacin. The incidence of teicoplanin reduced susceptibility revealed 32% without possessing either the vanA or vanB gene by the strains. PFGE revealed 56 well-defined genotypes indicating no clonal relationship of the strains. The propensity of S. haemolyticus to acquire resistance and its pathogenic potential in immunocompromised patients, especially among preterm neonates, emphasise the importance of species level identification of coagulase-negative staphylococci and routinely determine the MIC of proper antibacterial agents for these isolates.

Quantitative differences in antibiotic resistance between methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains isolated in Hungary, Austria and Macedonia.

The aim of this study was to compare the quantitative susceptibility of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA) strains from three European countries to nine antistaphylococcal agents. The antibiotic susceptibility of 274 MRSA and 284 MSSA strains from Hungary, Austria and macedonia was tested by the broth microdilution method. The clonal relationship of strains was determined by pulsed-field gel electrophoresis. Intermediate susceptibility to vancomycin appeared in Macedonian MRSA strains. Macedonian MRSA strains had high-level amikacin and gentamicin resistance. MSSA strains generally were susceptible to all drugs at minimum inhibitory concentrations (MIC(50)) except for gentamicin resistance in Macedonian strains. In Hungary and Austria a common antibiotic resistance phenotype of MRSA predominated, while in macedonia three other phenotypes were also prevalent. Geographical differences in the resistance of S. aureus are still high. Since resistance levels of MRSA and MSSA strains differ extensively, they should be considered separately for antibiotic resistance analysis.

Depressed activation of the lectin pathway of complement in hereditary angioedema.

The possibility of simultaneous measurement of the classical pathway (CP), mannan-binding lectin (MBL)--lectin pathway (LP) and alternative pathway (AP) of complement activation by the recently developed Wielisa method allowed us to investigate the in vivo significance of the C1-inhibitor (C1INH) in three complement activation pathways. Functional activity of the CP, LP and AP were measured in the sera of 68 adult patients with hereditary angioedema (HAE) and 64 healthy controls. In addition, the level of C1q, MBL, MBL-associated serine protease-2 (MASP-2), C4-, C3- and C1INH was measured by standard laboratory methods. MBL-2 genotypes were determined by polymerase chain reaction. Besides the complement alterations (low CP and C1INH activity, low C4-, C1INH concentrations), which characterize HAE, the level of MASP-2 was also lower (P = 0.0001) in patients compared with controls. Depressed LP activity was found in patients compared with controls (P = 0.0008) in homozygous carriers of the normal MBL genotype (A/A), but not in carriers of variant genotypes (A/O, O/O). Activity of CP correlated with LP in patients (Spearman's r = 0.64; P < 0.0001), but no significant correlation was found in the control group and no correlation with AP was observed. In contrast, the activity of CP and AP correlated (Spearman's r = 0.47; P < 0.0001) in healthy controls, but there was no significant correlation in the HAE patients. We conclude that the activation of LP might also occur in subjects with C1INH deficiency, which is reflected by the low MASP-2 and C4 levels.

Is MRSA more virulent than MSSA?

Numerous clinical studies have indicated, based on mortality rates, that methicillin-resistant Staphylococcus aureus (MRSA) strains are more virulent than methicillin-susceptible S. aureus (MSSA) strains. In contrast, quantitative laboratory examinations of the presence and magnitude of pathogenic mechanisms and virulence factors in strains of MRSA and MSSA have generated conflicting data. The most important reason for these conflicting results is probably the heterogeneic nature of the resistant population. A comparison of selected and congenic MRSA and MSSA sub-populations of the same strain is required to resolve this issue.

Extended-spectrum beta-lactamase-producing Klebsiella spp. in a neonatal intensive care unit: risk factors for the infection and the dynamics of the molecular epidemiology.

The extended-spectrum beta-lactamase (ESBL)-producing Klebsiella spp. cause worldwide problems in intensive care units. The aim of this study was to investigate the molecular epidemiology of ESBL-producing Klebsiella pneumoniae and K. oxytoca strains in a neonatal intensive care unit (NICU) in Budapest, Hungary and to determine the risk factors of the infections and the epidemiological features. Infections with Klebsiella spp. were analyzed retrospectively by reviewing the medical records between January 2001 and December 2005. Antibiotic susceptibility tests, isoelectric focusing, pulsed field gel electrophoresis, plasmid analysis, PCR for bla(TEM) and bla(SHV) and DNA sequencing analysis were performed on ESBL-producing Klebsiella isolates. A total of 45 babies were found to be infected with non-ESBL-producing Klebsiella spp. and 39 with ESBL-producing Klebsiella spp. Of the parameters analyzed, including sex, gestational age, twin pregnancy, birth weight, presence of central vascular catheter, mechanical ventilator use, parenteral nutrition, polymicrobial infection, caesarean section, transfusion and mortality, we found no statistically significant difference between the ESBL and the non-ESBL groups, or between the K. pneumoniae and K. oxytoca species. Further characterization of the ESBL-producing K. pneumoniae and K. oxytoca strains isolated between February 2001 and January 2003 revealed three distinct PFGE patterns of SHV-5-producing K. pneumoniae (A, B, E) and two distinct patterns of SHV-12-producing K. oxytoca (C,D) isolates; these had different plasmid profiles. From July to November 2005, a new SHV-5 producing K. oxytoca (F) was isolated. The molecular epidemiology of ESBL-producing organisms in a NICU over time shows substantial shifts in predominant strains. The ESBL production of the infected organisms has an impact on the survival of newborn babies with infections caused by Klebsiella spp.

The role of biological markers as predictors of response to preoperative chemotherapy in large primary breast cancer.

The aim of this prospective study was to evaluate biological markers, their correlation with response and outcome, and the change in these markers under the influence of preoperative chemotherapy (PCT) in patients with a large primary breast cancer. One hundred and thirty-five women were treated with PCT, followed by locoregional therapy and adjuvant treatment. Estrogen receptor (ER), progesterone receptor (PgR), HER-2, p53, and cathepsin D were determined by immunohistochemistry (IHC) before and after PCT. The overall response (OR) was 70% and the pathologic complete response (pCR) was 13%. Forty-four percent of the patients could be offered breast-conserving surgery (BCS). At a median follow-up of 50 mo the overall survival is 82% and the disease-free survival is 70%. No local recurrence (LR) has developed following BCS. Invasive ductal carcinoma (IDC) was more frequently ER-negative and HER-2-positive than invasive lobular carcinoma (ILC). P53-negative and ER-negative patients seemed to be more chemosensitive compared to p53-positive patients (74% vs 53%) and ER-positive patients (75% vs 65%), but this difference did not reach statistical significance. A trend toward higher complete pathologic remission rate was seen for ER-negative patients (p = 0.0609). PgR, HER-2, and cathepsin D were not related to response. The pattern of biological markers did not change with PCT, making repeated determination useless.

Better cosmetic results and comparable quality of life after skin-sparing mastectomy and immediate autologous breast reconstruction compared to breast conservative treatment.

Preoperative chemotherapy (PCT) can be used in large primary breast cancer to facilitate breast conservative surgery (BCS). Cosmetic results of BCS are influenced by the size of the residual tumour, relative to the size of the breast. After mastectomy, immediate breast reconstruction (IBR) with autologous tissue provides excellent cosmetic outcome and has proven to be safe in breast cancer patients. Besides improving overall and disease free survival, Quality of Life (QoL), body image and cosmetic outcome are also important issues after treatment for breast cancer. In this study, Health-Related-Quality of Life (HRQL) and body image were evaluated, in patients treated with PCT, followed by BCS, or skin-sparing mastectomy (SSM) and perforator-flap breast reconstruction. Additionally, clinical observers assessed cosmetic outcome. All participants were evaluated by the Medical Outcomes Study (MOS) 36-item Short Form Health Status Survey (SF-36, 36 items) and a study-specific questionnaire. An external panel evaluated standardised photographs of the breasts. For all patients, norm-based scores of physical and mental health state are comparable with the general population, except for vitality (VT) score, which is somewhat lower. No significant differences can be observed between both groups. The majority of the patients were satisfied with the appearance of their breasts. The cosmetic results, assessed by the clinical team, were significantly better for patients having IBR, compared to BCS. The mean score was 7.5/10 for IBR, versus 6.0/10 for BCS (p<0.0001).Breast conserving treatment or mastectomy with reconstruction may yield comparable results of QoL, but cosmetic outcome is better after SSM and perforator-flap reconstruction. Patients must be offered both options, and clinicians should stress that both are equally effective.

Inadvertent subdural injection: a complication of an epidural block.

Twenty-one hundred eighty two consecutive lumbar epidural injections were studied to determine the incidence of inadvertent subdural block retrospectively. A subdural block is defined as an extensive neural block in the absence of subarachnoid puncture, that is out of proportion to the amount of local anesthetic injected. Subdural injection is a complication of epidural block that probably occurs more frequently than previously recognized. An earlier report has estimated the incidence of subdural block to be 0.1%. This study, however, reports an incidence of 0.82% from a sample size of 2182 patients. Cadaveric dissection was also performed, further clarifying the presence and anatomic position of the subdural space.