Koch's bacillus - a look at the first isolate of Mycobacterium tuberculosis from a modern perspective.

Using molecular methods the authors have studied mycobacterial DNA taken from a 19th century victim of tuberculosis. This was the case from which Robert Koch first isolated and cultured the organism responsible for tuberculosis. The mycobacteria were preserved within five glass culture tubes as abundant bacterial colonies on slopes of a gelatinous culture medium of unknown composition. Originally presented by Koch to surgical laryngologist Walter Jobson Horne in London in 1901, the relic has, since 1983, been in the care of the Royal College of Surgeons of England. Light and electron microscopy established the presence of acid-fast mycobacteria but showed that morphological preservation was generally poor. Eleven different genomic loci were successfully amplified by PCR. This series of experiments confirmed that the organisms were indeed Mycobacterium tuberculosis and further showed that the original strain was in evolutionary terms similar to 'modern' isolates, having undergone the TB D1 deletion. Attempts to determine the genotypic group of the isolate were only partially successful, due in part to the degraded nature of the DNA and possibly also to a truncation in the katG gene, which formed part of the classification scheme. Spoligotyping resulted in amplification of DR spacers consistent with M. tuberculosis but with discrepancies between independent extracts, stressing the limitations of this typing method when applied to poorly preserved material.

Red cell traverse through thin glomerular basement membranes.

BACKGROUND: How red cells enter the urinary filtrate in most cases of hematuria of glomerular origin has remained a mystery despite the frequent ultrastructural examination of renal biopsy material. METHODS: Serial sections of glutaraldehyde-fixed, resin-embedded material from a case of sporadic microhematuria were examined by transmission electron microscopy when the site of a red cell traversing the glomerular capillary wall was fortuitously discovered on routine examination. RESULTS: The red cell assumed a dumbbell shape and traversed a localized gap 2.25 microm in diameter in the glomerular endothelium and basement membrane. Serial sections suggested a transcellular route. Apart from the thinning of the basement membrane (167 nm), there were no other generalized abnormalities. CONCLUSION: Red cells can traverse through gaps in the glomerular capillary walls to gain access to Bowman's space. This may be the origin of glomerular hematuria in common noninflammatory forms of glomerular disease, including thin basement membrane nephropathy.

Histological verification of computerised carotid plaque characterisation.

The correlation between the computerised measurement of carotid plaque echogenicity on high resolution ultrasound imaging and plaque histology was studied in 52 patients undergoing carotid endarterectomy. Thirteen plaques were from asymptomatic patients, 15 were associated with amaurosis fugax, 10 with transient ischaemic attacks and 14 with stroke. Longitudinal images of the anterior and posterior component of each plaque were obtained by ATL Ultramark-4 Duplex scanner and were transferred to a computer. Using an image analysis program the median of the overall grey scale content of each plaque component was evaluated and used as a measure of echogenicity. Following carotid endarterectomy each plaque specimen was divided into anterior and posterior component and then fixed, oriented, sectioned and stained in the longitudinal plane corresponding to the ultrasound image. Plaque histology sections were then examined by computer morphometric analysis and the percentage surface areas of fibrous tissue, lipid deposits and haemorrhage were calculated. This was then correlated with the grey scale median for each plaque component. Plaques with a high lipid and haemorrhage content as established histologically had a low grey scale median (Spearman correlation r=-0.351, p<0.05) and those with a high fibrous content had a high grey scale median (r=0.411, p<0.001). This study has shown that computerised measurement of carotid plaque echogenicity correlates well with histology and could be used to predict plaque composition, thus identifying high risk plaques with high lipid and haemorrhage content.

In situ hybridization studies of hepatitis A viral RNA in patients with acute hepatitis A.

In situ hybridization with oligonucleotide probes has been used to localise hepatitis A virus RNA genomic sequences in formalin-fixed and routinely processed human liver biopsies from three patients. Using radiolabelled Sulphur-35 antisense probes, viral genomic sequences were found in all three cases, but signal intensity was greatest in cases 1 and 2 with fulminant hepatitis, and was minimal in the third case of resolving hepatitis biopsied 2 months after acute illness. Localisation showed the viral RNA to be present in hepatocytes, sinusoidal cells and inflammatory cells in and around the portal tracts. Both cases showed signal in similar cell types, but the distribution of staining was predominantly periportal in case 1, whereas lobular staining was more apparent in case 2. Hybridization with sense polarity probes failed to detect any evidence of replicative intermediates of antigenomic viral RNA. The presence of hepatitis A RNA in phagocytic cells was confirmed using immunohistochemistry for a macrophage marker, CD68, combined with in situ hybridization. In all cases the signal was predominantly cytoplasmic, and this was confirmed with the use of tritiated probes.