Techniques of DNA-studies on prehispanic ectoparasites (Pulex sp., Pulicidae, Siphonaptera) from animal mummies of the Chiribaya Culture, Southern Peru

During a paleoparasitological survey of several animal mummies (Cavia aperea f. porcellus and Canis familiaris) from Chiribaya Baja, an archaeological site in Southern Peru, an unexpected find was made. In the well preserved fur, large numbers of mummified fleas (Pulex simulans/irritans)that parasitized the animals during life were encountered. Due to the relative recent event of the host mummification and the outstanding preservation of the fleas, an attempt for the retrieval of DNA was made. A DNA extraction and sequencing protocol for archaeological ectoparasitic remains has been established, taking additional studies for tissue and protein preservation into account. Tissue preservation was assessed with transmission electron microscopy and the protein preservation was tested through the racemisation ratios of aspartic acid. Regions of the 28S rDNA gene were successfully amplified and sequenced. Further research perspectives are outlined.

Stress radiography and posterior pathological laxity of knee: comparison between two different techniques.

Stress radiographs have been recommended in order to obtain a better objective quantification of abnormal compartment knee motion. This tool has showed to be superior in quantifying a posterior cruciate ligament (PCL) lesion compared to clinical or arthrometer evaluation. Different radiographic techniques have been described in literature to quantify posterior pathological laxity. In this study we evaluated the total amount of posterior displacement (PTD) and side to side difference (SSD), before and after surgical reconstruction of PCL or PCL and posterolateral complex (PLC), using two different stress radiography techniques (Telos stress and kneeling view). Twenty patients were included in this study. We found a statistical significant difference about both total PTD and SSD among the two techniques preoperatively and at follow-up, with greatest values occurring using the kneeling view. Although stress radiographies has been introduced to allow an objective quantification of laxity in ligamentous injured knee, we believe that further studies on a large numbers of subjects are required to define the relationship between PTD values, measured with stress knee radiography, particularly using kneeling view, and ligamentous knee injury, in order to obtain a real useful tool in the decision making process, as well as to evaluate the outcome after ligamentous surgery.

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques

Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and ethionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100%, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis.

New imaging techniques applied to Paleogene radiolaria

A simple and time efficient technique to illustrate specimens is described and demonstrated with Paleogene radiolarians. This method produces Scanning Electron Microscope (SEM) and composite focal depth Transmitted Light Microscope (TLM) images for single radiolarian specimens. We propose the use of this technique to clarify radiolarian taxonomy. This technique has distinct advantages over previously published time consuming techniques that can also require toxic materials.

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Cytostatic lung perfusion results in heterogeneous spatial regional blood flow and drug distribution: evaluation of different cytostatic lung perfusion techniques in a porcine model.

OBJECTIVES: Comparison of doxorubicin uptake, leakage and spatial regional blood flow, and drug distribution was made for antegrade, retrograde, combined antegrade and retrograde isolated lung perfusion, and pulmonary artery infusion by endovascular inflow occlusion (blood flow occlusion), as opposed to intravenous administration in a porcine model. METHODS: White pigs underwent single-pass lung perfusion with doxorubicin (320 mug/mL), labeled 99mTc-microspheres, and Indian ink. Visual assessment of the ink distribution and perfusion scintigraphy of the perfused lung was performed. 99mTc activity and doxorubicin levels were measured by gamma counting and high-performance liquid chromatography on 15 tissue samples from each perfused lung at predetermined localizations. RESULTS: Overall doxorubicin uptake in the perfused lung was significantly higher (P = .001) and the plasma concentration was significantly lower (P < .0001) after all isolated lung perfusion techniques, compared with intravenous administration, without differences between them. Pulmonary artery infusion (blood flow occlusion) showed an equally high doxorubicin uptake in the perfused lung but a higher systemic leakage than surgical isolated lung perfusion (P < .0001). The geometric coefficients of variation of the doxorubicin lung tissue levels were 175%, 279%, 226%, and 151% for antegrade, retrograde, combined antegrade and retrograde isolated lung perfusion, and pulmonary artery infusion by endovascular inflow occlusion (blood flow occlusion), respectively, compared with 51% for intravenous administration (P = .09). 99mTc activity measurements of the samples paralleled the doxorubicin level measurements, indicating a trend to a more heterogeneous spatial regional blood flow and drug distribution after isolated lung perfusion and blood flow occlusion compared with intravenous administration. CONCLUSIONS: Cytostatic lung perfusion results in a high overall doxorubicin uptake, which is, however, heterogeneously distributed within the perfused lung.

Techniques for CT and MR, post-processing, radiation

Brain perfusion can be assessed by CT and MR. For CT, two major techniques are used. First, Xenon CT is an equilibrium technique based on a freely diffusible tracer. First pass of iodinated contrast injected intravenously is a second method, more widely available. Both methods are proven to be robust and quantitative, thanks to the linear relationship between contrast concentration and x-ray attenuation. For the CT methods, concern regarding x-ray doses delivered to the patients need to be addressed. MR is also able to assess brain perfusion using the first pass of gadolinium based contrast agent injected intravenously. This method has to be considered as a semi-quantitative because of the non linear relationship between contrast concentration and MR signal changes. Arterial spin labeling is another MR method assessing brain perfusion without injection of contrast. In such case, the blood flow in the carotids is magnetically labelled by an external radiofrequency pulse and observed during its first pass through the brain. Each of this various CT and MR techniques have advantages and limits that will be illustrated and summarized.Learning Objectives:1. To understand and compare the different techniques for brain perfusion imaging.2. To learn about the methods of acquisition and post-processing of brain perfusion by first pass of contrast agent for CT and MR.3. To learn about non contrast MR methods (arterial spin labelling).

The promise of advanced imaging techniques for the detection of hepatitis C virus antigens in the infected liver.

Injection drug use before and after liver transplantation: a retrospective multicenter analysis on incidence and outcome. Clin Transplant 2009 DOI: 10.1111/j.1399-0012.2009.01121.x. Background and aims: Injecting drug use (IDU) before and after liver transplantation (LT) is poorly described. The aim of this study was to quantify relapse and survival in this population and to describe the causes of mortality after LT. Methods: Past injection drug users were identified from the LT listing protocols from four centers in Switzerland and France. Data on survival and relapse were collected and used for uni- and multivariate analysis. Results: Between 1988 and 2006, we identified 59 patients with a past history of IDU. The mean age at transplantation was 42.4 yr and the majority of patients were men (84.7%). The indication for LT was for the vast majority viral cirrhosis accounting for 91.5% of cases, while alcoholic cirrhosis was 5.1%. There were 16.9% of patients who had a substitution therapy before and 6.8% who continued after LT. Two patients (3.4%) relapsed into IDU after LT and died at 18 and 41 months. The mean follow-up was 51 months. Overall survival was 84%, 66%, and 61% at 1, 5, and 10 yr after transplantation. Conclusions: Documented IDU was rare in liver transplanted patients. Past IDU was not associated with poorer survival after LT, and relapse after LT occurred in 3.4%.

IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB), we performed IS6110-based restriction fragment length polymorphism analysis of strains, isolated from 105 patients during the period of September 2002 to March 2003 in TB centers and university hospitals of the province. Among 105 isolates, 81 different IS6110 patterns were found, of which 70 were observed only once and 11 were shared by two to eight isolates. Ninety-six isolates (91.4%) were found to have more than five copies of IS6110 and together with high patterns polymorphism, shows that IS6110-RFLP typing could be useful for studying the epidemiology of TB in Azerbaijan. The minimum estimated rate of recent transmission was 23%, suggesting that the degree of recent transmission in East Azerbaijan Province is relatively low. Clustering was not associated with age, sex or site of infection of TB but drug-resistant isolates were less likely to be clustered than sensitive isolates (p < 0.05).

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization

Typing of Mycobacterium tuberculosis strains isolated in Community Health Centers of Rio de Janeiro City, Brazil

Fingerprinting of Mycobacterium tuberculosis strains from tuberculosis (TB) patients attended in Community Health Centers (CHCs) of Rio de Janeiro was performed to verify possible risk factors for TB transmission. A prospective community-based study was performed during the period of July 1996 to December 1996 by collecting sputum samples of 489 patients in 11 different CHCs in four different planning areas (APs) of the city. Bacteriological, clinical, and epidemiological information was collected and M. tuberculosis genotypes defined after restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element (DRE) fingerprinting of RFLP-clustered cases. Risk factors for TB transmission were looked for using three levels of cluster stringency. Among 349 (71%) positive cultures obtained, IS6110-RFLP typing could be performed on strains from 153 different patients. When using identity of RFLP patterns as cluster definition, 49 (32%) of the strains belonged to a cluster and none of the clinical or epidemiologic characteristics was associated with higher clustering levels. However, higher clustering level was observed in the AP including the central region of the city when compared to others. This strongly suggests that more recent transmission occurs in that area and this may be related with higher incidence of TB and HIV in this region.