A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy

Permeabilising electric pulses can be advantageously used for DNA electrotransfer in vivo for gene therapy, as well as for drug delivery. In both cases, it is essential to know the electric field distribution in the tissues: the targeted tissue must be submitted to electric field intensities above the reversible permeabilisation threshold (to actually permeabilise it) and below the irreversible permeabilisation threshold (to avoid toxic effects of the electric pulses). A three-dimensional finite element model was built. Needle electrodes of different diameters were modelled by applying appropriate boundary conditions in corresponding grid points of the model. The observations resulting from the numerical calculations, like the electric field distribution dependence on the diameter of the electrodes, were confirmed in appropriate experiments in rabbit liver tissue. The agreement between numerical predictions and experimental observations validated our model. Then it was possible to make the first precise determination of the magnitude of the electric field intensity for reversible (362+/-21 V/cm, mean +/- S.D.) and for irreversible (637+/-43 V/cm) permeabilisation thresholds of rabbit liver tissue in vivo. Therefore the maximum of induced transmembrane potential difference in a single cell of the rabbit liver tissue can be estimated to be 394+/-75 and 694+/-136 mV, respectively, for reversible and irreversible electroporation threshold. These results carry important practical implications.     

Bioactivities of seven chrysanthemum species flowers powder on Spodoptera littoralis (Boiduval) larvae

We have screened seven Chrysanthemum species, collected from different biotope in Tunisia for the antifeeding and growth regulatory activity against S. littoralis (Lepidoptera: Noctuidae) an insect pest of tomato. The flowers powder was added to the Poitout and Bues (1974) artificial diet at three concentrations: 4, 8, and 16%. After ten days of treatment, the consumed quantity determination revelled that these species have an antifeeding activity proportional to the concentration, more considerable for the C. fuscatum and C. Myconis flowers powder. At the concentration of 16% the larvae weight was significantly delayed in the average time to the control, these results were compared to the effect of Cestrum parquii on S. littoralis and Pieris brassicae The moulting date determination showed an elongation of the third, fourth and fifth stages at the high concentration probably due to the presence of juvenile hormones Analogues.     

Cytogenetic survey of 117 Tunisian patients with de novo myelodysplastic syndrome

Cytogenetic studies were performed on 117 Tunisian patients with de novo myelodysplastic syndromes (MDS). According to the French-American-British (FAB) criteria 40 patients presented with refractory anaemia (RA, 34%), eight with refractory anaemia with ringed sideroblasts (RARAS, 7%), 19 with refractory anaemia with excess of blasts (RAEB, 16%), 16 with refractory anaemia with excess of blasts in transformation (RAEB-t, 14%), 18 had chronic myelomonocytic leukaemia (CMML, 15%) and 16 unclassifiable MDS (14%). Seventy-five were men and forty-two were women. Five were children and 112 were adults with a median age of 58 years. Fifty-five per cent of the patients presented clonal chromosome abnormalities. Rates of abnormality varied from one FAB subtype to the other: 55% in RA, 75% in RARAS, 63% in RAEB, 75% in RAEB-t and 28% in CMML. The most frequent chromosome abnormalities were del(5q) (22 cases), monosomy 7 (12 cases), del(12p) (6 cases), and trisomy 8 (5 cases). Rare abnormalities were also found: ring of chromosome 12 and trisomy 15. Conventional cytogenetics remains the basic technique in identifying chromosomal abnormalities associated with MDS.     

Cytogenetic analysis in 139 Tunisian patients with de novo acute myeloid leukemia

This paper presents the results of a cytogenetic analysis in 139 Tunisian patients with de novo acute myeloid leukemia (AML), including 27 children aged 1-15 years and 112 adults. Mean age was 32 (range 1-75) and the M/F ratio was 1.43. Of our patients, 45% had apparently normal karyotypes. Acquired chromosome aberrations were found in 77 (55% ) patients. t(8;21) was identified in 27 patients (19%); t(15;17) in 13 patients (9%); deletion 7q or monosomy 7 in seven patients (5%); +8 in seven patients (5%); abnormal 16 in four patients (3%); 11q23 rearrangements in two patients (2%) and del(5q), in one patient (1%). The remaining 16 patients had miscellaneous clonal abnormalities. Specific translocations associated with the FAB type were found: t(8;21) with AML2 and t(15;17) with AML3. We concluded that our study in a Tunisian population confirmed the relation between some specific abnormalities and the FAB classification. We found a higher incidence for t(8;21) than usually described.     

A Patient-Centered Understanding of the Referral System in Ethiopian Primary Health Care Units

Background

Primary healthcare systems in sub-Saharan Africa have undergone substantial development in an effort to expand access to appropriate facilities through a well-functioning referral system. The objective of this study was to evaluate the current patterns of seeking prior care before arriving at a health center or a hospital as a key aspect of the referral system of the primary health care unit (PHCU) in three regions in Ethiopia. We examined what percentage of patients had either sought prior care or had been referred to the present facility and identified demographic and clinical factors associated with having sought prior care or having been referred.

Methods and Findings

We conducted a cross-sectional study using face-to-face interviews in the local language with 796 people (99% response rate) seeking outpatient care in three primary health care units serving approximately 100,000 people each and reflecting regional and ethnic diversity; 53% (N = 418) of the sample was seeking care at hospital outpatient departments, and 47% of the sample was seeking care at health centers (N = 378). We used unadjusted and adjusted logistic regression to identify factors associated with having been referred or sought prior care. Our findings indicated that only 10% of all patients interviewed had been referred to their current place of care. Among those in the hospital population, 14% had been referred; among those in the health center population, only 6% had been referred. Of those who had been referred to the hospital, most (74%) had been referred by a health center. Among those who were referred to the health center, the plurality portion (32%) came from a nearby hospital (most commonly for continued HIV treatment or early childhood vaccinations); only 18% had come from a health post. Among patients who had not been formally referred, an additional 25% in the hospital sample and 10% in the health center sample had accessed some prior source of care for their present health concern. In the adjusted analysis, living a longer distance from the source of care and needing more specialized care were correlated with having sought prior care in the hospital sample. We found no factors significantly associated with having sought prior care in the health center sample.

Conclusions

The referral system among health facilities in Ethiopia is used by a minority of patients, suggesting that intended connections between health posts, health centers, and hospitals may need strengthening to increase the efficiency of primary care nationally.     

Evaluation of Two Influenza Surveillance Systems in South Africa

Background

The World Health Organisation recommends outpatient influenza-like illness (ILI) and inpatient severe acute respiratory illness (SARI) surveillance. We evaluated two influenza surveillance systems in South Africa: one for ILI and another for SARI.

Methodology

The Viral Watch (VW) programme has collected virological influenza surveillance data voluntarily from patients with ILI since 1984 in private and public clinics in all 9 South African provinces. The SARI surveillance programme has collected epidemiological and virological influenza surveillance data since 2009 in public hospitals in 4 provinces by dedicated personnel. We compared nine surveillance system attributes from 2009–2012.

Results

We analysed data from 18,293 SARI patients and 9,104 ILI patients. The annual proportion of samples testing positive for influenza was higher for VW (mean 41%) than SARI (mean 8%) and generally exceeded the seasonal threshold from May to September (VW: weeks 21–40; SARI: weeks 23–39). Data quality was a major strength of SARI (most data completion measures >90%; adherence to definitions: 88–89%) and a relative weakness of the VW programme (62% of forms complete, with limited epidemiologic data collected; adherence to definitions: 65–82%). Timeliness was a relative strength of both systems (e.g. both collected >93% of all respiratory specimens within 7 days of symptom onset). ILI surveillance was more nationally representative, financially sustainable and expandable than the SARI system. Though the SARI programme is not nationally representative, the high quality and detail of SARI data collection sheds light on the local burden and epidemiology of severe influenza-associated disease.

Conclusions

To best monitor influenza in South Africa, we propose that both ILI and SARI should be under surveillance. Improving ILI surveillance will require better quality and more systematic data collection, and SARI surveillance should be expanded to be more nationally representative, even if this requires scaling back on information gathered.     

Carnitine and acylcarnitine profiles in dried blood spots of patients with acute myocardial infarction

Earlier studies have suggested an important role of carnitine pathway in cardiovascular pathology. However, the redistribution of carnitine and acylcarnitine pools, as a result of altered carnitine metabolism, is not clearly known in patients with acute myocardial infarction (AMI). We compared the carnitine and acylcarnitine profiles of 65 AMI patients, including 26 ST-elevated myocardial infarction (STEMI) and 39 non-ST-elevated myocardial infarction (NSTEMI), 28 patients with chest pain and 154 normal controls. The levels of carnitine and acylcarnitines in the blood spots were determined using LC-MS/MS. Total and free carnitine levels were significantly higher in all the patient groups in the following order: STEMI > NSTEMI > chest pain. The levels of short- and medium-chain acylcarnitines were significantly higher in patient groups. Among the long-chain acylcarnitines, C14:2 and C16:1 levels were significantly increased in STEMI and NSTEMI. The ratio of free carnitine to short-chain or medium-chain acylcarnitines was significantly decreased in STEMI, NSTEMI and chest pain patients however a significant increase was observed in the ratio of carnitine to long-chain acylcarnitines in all the patient groups as compared to normal controls. In conclusion, alterations in carnitine and acylcarnitine levels in the blood of AMI patients indicate the possibility of impaired carnitine homeostasis in ischemic myocardium. The clinical implications of these findings for the risk screening or diagnosis and prognosis of AMI require additional follow-up studies on large number of patients. We also suggest that a dual-marker strategy using carnitine (longer plasma half-life) in combination with troponin (shorter plasma half-life) could be a more promising biomarker strategy in risk stratification of patients.     

Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants

Changes in the cell envelope composition of mycobacteria cause major changes in cytokine profiles of infected antigen presenting cells. We describe here the modulation of inflammatory responses by Mycobacterium abscessus, an emerging pathogen in cystic fibrosis. M. abscessus is able to switch from a smooth (S) to a rough (R) morphotype by the loss of a surface glycopeptidolipid. R variants are associated with severe clinical forms and a 'hyper-proinflammatory' response in ex vivo and in vivo models. Using partitioning of cell surface components we found that a complex fraction, more abundant in R variants than in S variants, made a major contribution to the TLR-2-dependent hyper-proinflammatory response induced by R variants. Lipoproteins were the main TLR-2 agonists in this fraction, consistent with the larger amounts of 16 lipoproteins in cell surface extracts from R variants; 15 out of 16 being more strongly induced in R variant than in S variant. Genetic interruption of glycopeptidolipid pathway in wild-type S variant resulted in R phenotype with similar induction of lipoprotein genes. In conclusion, R morphotype in M. abscessus is associated with increased synthesis/exposure at the cell surface of lipoproteins, these changes profoundly modifying the innate immune response through TLR-2-dependent mechanisms.     

Analytical Performance of the Roche LightCycler? Mycobacterium Detection Kit for the Diagnosis of Clinically Important Mycobacterial Species

Background

The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility.

Methodology/Principal Findings

Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay''s reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent.

Significance

The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.