Simple and rapid differentiation of Mycobacterium tuberculosis H37Ra from M. tuberculosis clinical isolates through two cytochemical tests using neutral red and nile blue stains

The attenuated Mycobacterium tuberculosis strain H37Ra is one of the most commonly used controls for M. tuberculosis identification in the clinical laboratory and is a source of false-positive results for M. tuberculosis as a consequence of cross-contamination. Therefore, the ability to discriminate between H37Ra and real clinical isolates has important public health implications. To date, differentiation of H37Ra from M. tuberculosis clinical isolates is possible only by IS6110 genotyping and spoligotyping. In the 1950s, some authors reported that the virulent strain H37Rv and M. tuberculosis clinical isolates were able to fix basic dyes in their anionic forms, while H37Ra was not. We have studied the different techniques described for M. tuberculosis cytochemical staining and have chosen the best of these, introducing certain modifications in order to increase their discriminative power and reproducibility. We describe cytochemical staining of M. tuberculosis cells with neutral red and Nile blue, which differentiates H37Ra from virulent strains. This method could be used as an easy laboratory tool for distinguishing between H37Ra and real M. tuberculosis clinical isolates.     

Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology

3‐Methylglutaconic aciduria (3‐MGA‐uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole‐exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3‐MGA‐uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High‐resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50‐deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild‐type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.     

Aortic intramural hematoma during coronary angioplasty: insights into the pathogenesis of intramedial hemorrhage.

We report a case of a 74-year-old woman who had an aortic intramural hematoma as a complication of percutaneous coronary angioplasty. Transesophageal echocardiography enabled the diagnosis of aortic intramural hematoma and was very useful in the patient's management and follow-up.     

Nuria Durany i Pich (1961–2010)

Obituary

Nuria Durany i Pich (1961–2010)     

Usefulness of a Lung Cancer Rapid Diagnosis Specialist Clinic. Contribution of Ultrasound Bronchoscopy

ObjectiveTo analyse the results obtained in the diagnosis and staging of lung cancer (LC) by a Lung Cancer Rapid Diagnosis Unit (LC-RDU) in which real-time endobronchial ultrasound-guided transbronchial needle aspiration (RT-EBUS guided-TBNA) is performed as part of the clinical evaluation of the patient prior to treatment.MethodA four year observational study was conducted on a group of patients evaluated due to suspicion of LC in an LC-RDU. The times and the techniques required for the diagnosis, the treatment period and the level of the disease in the initial staging were recorded.ResultsOut of a total of 678 patients seen in the LC-RDU, the diagnosis in 352 was confirmed in one or more histopathology tests. In 170 patients (48.2%) the diagnosis was made with biopsies and/ or cytology samples obtained by fibrobronchoscopy, and RT-EBUS guided-TBNA confirmed the clinical suspicion in 70 patients (19.9%). In the 280 patients with NSCLC, 166 RT-EBUS guided-TBNA were performed for staging (59.3%), and in 105 of them the technique only showed local disease (37.5%). Eighty-three of these patients underwent therapeutic surgery, which was radical in 73 cases (87.9%).ConclusionIn half of the patients referred to the LC-RDU due to suspected LC, the diagnosis was confirmed in 75% of cases using endoscopic techniques. RT-EBUS guided-TBNA, which was the diagnostic technique in 20% of the cases and for staging in more than half of them, led to reduced waiting times to diagnosis and onset to treatment.     

Role of intraoperative echo-Doppler color echocardiography in cardiac surgery. Preliminary study

Intraoperative echocardiography with Doppler color flow imaging technique can provide the surgeon with an immediate and direct assessment of cardiac anatomy and function. To determine the utility of this recent technique, we have examined 15 patients pre, per, and postoperatively with Doppler color flow imaging. The intraoperative study was performed before and after cardiopulmonary bypass. A sterile transducer was placed directly on the epicardial surface of the heart, and multiple views were recorded. Intraoperative echocardiography with Doppler color flow imaging technique demonstrated excellent correlation with preoperative and postoperative findings, allowing an immediate evaluation of the surgical results. These preliminary results suggest that intraoperative Doppler color flow imaging technique is a valuable adjunct in heart surgery. It is particularly useful in valve repair, and in surgical repair of congenital heart disease.     

Localization of the neuronal antigen recognized by anti-Tr antibodies from patients with paraneoplastic cerebellar degeneration and Hodgkin’s disease in the rat nervous system

Patients with paraneoplastic cerebellar degeneration and Hodgkin’s disease develop autoantibodies (Tr-Ab) that immunoreact with the cytoplasm of the Purkinje cells and produce a characteristic punctate pattern in the molecular layer of the cerebellum. In the present study, we analyzed the structures of the adult rat cerebellar cortex identified by Tr-Ab and the expression of the antigen recognized by Tr-Ab in the developing rat brain. By laser confocal microscopy and immunoelectron microscopy, Tr-Ab immunoreactivity was found localized in the cytosol and outer surface of the endoplasmic reticulum of the perikarya of neurons of the molecular layer and the cell body and dendrites of Purkinje cells without a particular concentration in dendritic spines. Tr-Ab reactivity was more widespread in the developing rat brain. Tr-Ab labeling of Purkinje cells was already observed at P0 (day of birth). The staining of the molecular layer followed the development of the dendritic tree. The internal and inner level of the external granule cell layer were labeled with Tr-Ab with a dotted pattern that became almost negative by the 2nd postnatal week. The staining probably corresponded to granule cells as suggested by the positive Tr-Ab labeling of cultures of embryonic granule neurons. The present findings suggest that the antigen recognized by Tr-Ab appears early and is widely expressed in the developing rat brain. In the adult cerebellum, the antigen is localized in the cell body and dendrites of the Purkinje cells but is not concentrated in the dendritic spines. Received: 28 October 1997 / Revised, accepted: 16 December 1997