Rapid identification by the Micro-ID system of Enterobacteriaceae detected by urine screening.

Previous studies have demonstrated that organisms detected by urine screening can be processed for rapid identification and antimicrobial susceptibility testing directly from urine or urine screening broth. In the present study, an improved method for processing such specimens was evaluated. Organisms were harvested by centrifugation from positive urine screening broth, and inocula were prepared for rapid identification by the Micro-ID system and rapid susceptibility testing by the Autobac system. Nearly 2,500 urine specimens were analyzed by urine screening, and 206 specimens had significant growth of gram-negative, oxidase-negative bacilli. These organisms, prepared by the centrifugation procedure, were identified and tested for susceptibility to antimicrobial agents. For comparison, identifications by the Micro-ID system and antimicrobial susceptibility tests by the Autobac system were performed on the same organisms the next day with inocula prepared from colonies growing from standard urine cultures. The results demonstrated that 95% of the organisms were correctly identified by this procedure, and susceptibility testing by the rapid method gave results in 94% agreement with the standard method. These results demonstrate that organisms detected by urine screening can be accurately identified and tested for antimicrobial susceptibility after centrifugation from urine screening broth. This system provides a practical procedure or same-day reporting of urine culture results.     

Specificity and mechanism of the histone methyltransferase Pr-Set7

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.     

Expression of Ki-67 and cytokeratin 20 in hyperplastic polyps of the colorectum

AIMS: To study the expression of Ki-67 and cytokeratin 20 (CK20) in a group of hyperplastic polyps (including a group with "atypical" features) with the aim of determining whether upper crypt Ki-67 staining and lower crypt CK20 staining correlated with these atypical features, as assessed by light microscopy. METHODS: Fifty seven formalin fixed, paraffin wax embedded hyperplastic colorectal polyps from 53 patients were selected on histological grounds; these comprised 26 typical polyps and 31 with atypical features, which included nuclear hyperchromatism, basal crowding, and increased mitotic activity. These polyps were examined using a standard immunohistochemical method with antibodies against CK20 and Ki-67. Comparisons were made with normal mucosa, adenomatous polyps, and carcinomas. RESULTS: Of the 26 typical polyps, 17 showed the usual pattern of lower crypt Ki-67 and upper crypt CK20 staining; one with upper crypt Ki-67 staining but normal surface CK20 staining; seven with Ki-67 confined to the lower half of crypts but with scattered lower crypt CK20; and one with both upper crypt Ki-67 staining, together with scattered CK20 basal staining. Of the 31 polyps with atypical features, 11 showed the usual staining pattern of lower crypt Ki-67 staining and surface staining with CK20; two showed Ki-67 staining extending into the upper half of crypts, but with a normal surface staining with CK20; 14 showed Ki-67 confined to the lower half of crypts, but scattered lower crypt staining with CK20; and four showed upper crypt Ki-67 staining together with scattered CK20 lower crypt staining. CONCLUSIONS: The normal pattern of lower crypt Ki-67 and upper crypt CK20 was seen in 28 of the 57 hyperplastic polyps and, in general, this corresponded with standard light microscopic appearances. Twenty one of the 57 polyps showed lower crypt mosaic CK20 staining, which in general corresponded with basal abnormalities on light microscopy, although seven specimens had normal appearances. Two smaller subsets emerged, one showing upper crypt Ki-67 staining in the presence of normal CK20 expression (three cases) and another in which a combination of lower crypt CK20 and upper crypt Ki-67 expression was seen (five cases). This last pattern was similar to that of neoplastic polyps and raises the possibility that a subgroup of hyperplastic polyps exists that may be a variant with malignant potential. Further studies with markers of mismatch repair genes and K-ras mutations may help to clarify this issue.     

Pharmacokinetics of CAMPATH-1H: assay development and validation

CAMPATH-1H is a humanised monoclonal antibody against the CD52 antigen which is being developed for treatment of chronic lymphocytic leukaemia (CLL), autoimmune disease and prevention of transplant rejection. Measurement of antibody serum levels is important for optimising dose regimens but difficult owing to the low concentration compared with normal human IgG.

After consideration of various methods, a suitable assay was developed based on indirect immunofluorescence. Test samples were incubated with target cells (HUT-78, a human T cell line) and the CAMPATH-1H was detected by binding of a fluorescent-labelled anti-human Ig using a flow cytometer. Robustness of the assay was demonstrated under a range of experimental conditions. Because of the low affinity of CAMPATH-1H, only a weak signal was seen at low concentrations. The limit of detection was 0.15 μg/ml and the limit of quantitation was 0.25 μg/ml. Since serum samples were diluted at least 1:2, the lowest concentration which can be measured in patient serum was 0.5 μg/ml. The overall precision (coefficient of variation) was ±13% and the overall accuracy (bias) was +9%. There was a low incidence of false-positive results (<2%) in normal or pre-treatment patient serum. Quantitative recovery was obtained from serum samples spiked with CAMPATH-1H and stored under a variety of conditions, including being treated at 56 °C for 30 min and frozen and thawed up to four times.

This validated assay is suitable for the measurement of CAMPATH-1H levels in clinical trials and the same principles may be applied to any other cell-binding monoclonal antibody.     

Rapid Semiautomated Screening and Processing of Urine Specimens

A rapid urine culture procedure was evaluated in which positive urines were detected by using light-scatter photometry (Autobac). Specimens were analyzed at 3, 5, and 6 h. Specimens detected as positive at 3 h were then further evaluated by a direct 3-h susceptibility procedure (Autobac) and by a 4-h identification procedure (Micro-ID). Of 949 specimens, 175 had >10⁵ colony-forming units per ml by colony count. Of these latter specimens, 75.4% had been detected by 3 h, and 95.4% were detected by 6 h. Of specimens positive by Autobac at 3 h, 96% (95.7%) had >10⁵ colony-forming units per ml. If pure by Gram stain, those positive specimens were inoculated to direct susceptibility and identification systems. When direct Autobac susceptibilities were compared with the standard Autobac method done from the plate the following day, discrepancy rates were 1.3% very major, 2.1% major, and 7.4% total. The direct identifications were 94% (94.2%) correct when using the Micro-ID manual and a collection of octal patterns unique to this system, in which urine/broth culture inoculum was employed instead of the usual organism colony suspension. Those urine specimens negative after screening at 3 h were evaluated at 5 and 6 h, and an additional 126 specimens were detected as positive. These were then processed by routine plate inoculation, due to the limitations of the work day. By 6 h, 95.4% of specimens with >10⁵ colony-forming units per ml were detected. The 4.6% false-negative results consisted of patients on antibiotics, or slowly growing bacteria suspected of being distal urethral contaminants. Thus, 83.5% of the urine cultures received by 9:00 a.m. (10.6% 3-h positives and 72.9% negative at 6 h) could be evaluated and reported within one 8-h work day.     

Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri.

A 140-megadalton plasmid (pWR110), which has previously been associated with virulence in Shigella flexneri, was transferred to Escherichia coli K-12. Segments of S. flexneri chromosomal material were then transferred to the plamid-bearing K-12 strains. The virulence of these transconjugant hybrids was assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test. A K-12 strain which harbored only pWR110 invaded HeLa cells, produced minimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test. Plasmid-containing K-12 hybrids which had incorporated various shigella chromosomal regions gave differential reactions in the rabbit ileal loops and in the Sereny test. Analysis of these transconjugants indicated that three regions were linked with virulent phenotypes. These included the his region (when the genes responsible for O-antigen synthesis were cotransferred) and the kcp locus (linked to the lac-gal region). Either of these chromosomal regions was sufficient to allow invasion of the rabbit ileal mucosa. In addition to both of these regions, another shigella chromosomal segment linked to the arg and mtl loci was necessary for fluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus, derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transfer of a large plasmid and three chromosomal segments from S. flexneri, appeared to contain the necessary determinants for full pathogenicity in a variety of laboratory models.     

Telepathology for routine light microscopic and frozen section diagnosis

Telepathology (TP) uses telecommunication linkages to electronically capture, store, retrieve, and transmit images to distant sites. We assessed the feasibility of a dynamic real-time TP system for light microscopic (LM) diagnosis of anatomic pathology specimens, including frozen sections. Six pathologists, in 2 separate periods, read a set of 160 retrospectively retrieved slides (80 of which were frozen sections) by TP and LM. Reading times were recorded. Diagnoses were compared with the reference diagnosis (established by a group of 5 independent pathologists) and graded on a scale of 0 to 2 (2, correct; 1, incorrect but no clinical impact; 0, incorrect with clinical impact). Overall, LM was more accurate than TP compared with the reference diagnosis (score, 1.68 vs 1.54). There was no difference in accuracy between frozen section and paraffin-embedded tissue. Intraobserver agreement ranged from 82.5% to 88.2%. The average reading time was 6.0 minutes for TP and 1.4 minutes for LM. During the study, reading time decreased for TP but not for LM. These results show that despite marginally lower accuracy and longer reading times, TP isfeasible for routine light microscopic diagnosis, including frozen sections.