Ellipticine derivative induces potent cytostatic effect in acute myeloid leukaemia cells.

A panel of novel ellipticine isomers were designed and synthesised with the aim of evaluating their anti-cancer effects on selected leukaemia cell lines. A preliminary NCI 60-cell screen demonstrated that these compounds display promising anti-tumour activity across a number of different cell types. We have consequently examined the effect of these derivatives in detail on the Acute Myeloid Leukaemia (AML) cell line, MV4-11. Cell cycle analyses revealed that the compounds had a range of distinctive cell cycle effects. 7-Hydroxyisoellipticine showed the most promise with respect to cytostatic activity. We demonstrated that this compound inhibited proliferation of leukaemia cells by preventing cells from progressing from G2 phase into mitosis over a period of 24 h at a concentration of 5 µM. Our research suggests that this is mediated by an induction of reactive oxygen species (ROS), which in turn activates the DNA damage response pathway. As a result of the activation of p53, cyclin B1 is inhibited. The induction of this pathway leads to apoptosis which is seen at 48 h using the same dose of 7-hydroxyisoellipticine. This study provides for the first time detailed cellular information on the potential use of isoellipticines as chemotherapeutic agents.

Use of a dry latex agglutination test to measure eradication of Helicobacter pylori infection.

BACKGROUND AND METHODS: A total antibody latex serology test was compared with enzyme immunoassay serology after treatment for Helicobacter pylori infection in 22 patients. RESULTS: Nineteen patients were cured of infection, but only nine (47%) were negative by the latex test after 6 months. However a significant decline in immunoglobulin (Ig)G was seen in 90% of the cured patients. CONCLUSIONS: Although the latex test is suitable for initial diagnosis of H. pylori infection, it is not suitable for monitoring treatment success. A decline in IgG of more than 40% correlates well with successful eradication of H. pylori.

Evaluation of two serological tests for the diagnosis of chlamydial respiratory disease.

Serological tests for chlamydial infection are one of the most frequently used methods in the diagnosis of atypical respiratory infections. Use of serological tests has implicated chlamydial infections in asthma, arthritis and coronary heart disease, but the specificity of chlamydial serology tests has been questioned. The immunofluorescence test is the most sensitive and specific serological test available for detection of chlamydial antibodies. This study compares two commercially available immunofluorescent antibody tests. The SeroFIA test using purified elementary bodies of Chlamydia pneumoniae, C. psittaci and C. trachomatis, detected 24 cases of acute C. pneumoniae infection, whereas the Spot IF test using whole cell antigen of C. psittaci and C. trachomatis, misdiagnosed 20 of these as psittacosis and missed four cases.

Selection of optimum laboratory tests for the identification of Moraxella catarrhalis.

We evaluated a variety of conventional and rapid tests and examined the erythromycin susceptibility of a collection of Moraxella catarrhalis and commensal neisseria strains in order to determine the optimum method for routine identification. One hundred and fifty three strains were tested by Gram stain, catalase, oxidase, carbohydrate degradation by two methods and the presence of esterases using indoxyl acetate, 4-methylumbelliferyl butyrate (MUB). Tween 80 and tributyrin as substrates. Erythromycin MICs and zone diameters around 1, 5 and 15 micrograms discs were determined by the NCCLS method for 151 of the strains. A combination of Gram stain, oxidase and either indoxyl acetate, spot MUB or tributyrin hydrolysis test proved to be reliable and potentially the most convenient for routine testing. MICs and zone diameters easily distinguished between the erythromycin-sensitive M. catarrhalis and the erythromycin-resistant commensal neisserias and would provide confirmation of identification if used for susceptibility testing.

Investigation of an apparent cluster of Klebsiella pneumoniae bacteremias using random amplified polymorphic DNA analysis.

A cluster of bacteremia episodes with Klebsiella pneumoniae was noted in patients in a hematology-oncology ward during a 3-week period. Random amplified polymorphic DNA (RAPD) analysis, a novel technique for generating chromosomal fingerprints from bacterial isolates, was used as an aid to the epidemiological investigation of this cluster. For each of the two patients from whom multiple isolates had been obtained, identical RAPD patterns were observed in the serial isolates, even for a patient where the isolates had different biotypes. Isolates from different patients gave distinct patterns. Random amplified polymorphic DNA was found to be a useful typing technique for this cluster of K pneumonia bacteremias.

The WHO multicenter study on Listeria monocytogenes subtyping: random amplification of polymorphic DNA (RAPD).

As part of a WHO multicenter study on Listeria monocytogenes subtyping methods the random amplification of polymorphic DNA (RAPD)-technique was evaluated. Six participants were asked to use a standard protocol to analyse a set of 80 L. monocytogenes strains. This set contained 22 groups of epidemiologically linked isolates and 11 pairs of duplicate strains. Using three different 10-mer primers the median reproducibility of the RAPD-results obtained by the six participants was 86.5% (range 0-100%). Failure in reproducibility was mainly due to results obtained with one particular primer. The number of epidemiological groups found to be homogeneous varied from 1-22 (median 16). However, for some groups an inhomogeneity was found by the majority of participants. The overall correlation between the results from the different participants ranged from 32 to 85%.

A practical single sample dry latex agglutination test for Helicobacter pylori antibody detection.

Assessment of a single serum sample for Helicobacter pylori antibodies is frequently requested in routine diagnostic laboratories. Current enzyme linked immunosorbent assay (ELISA) kits are not ideal for testing small numbers of serum samples and some have low sensitivities, specificities or large grey zones. A panel of 90 serum samples from patients who had presented for routine upper endoscopy was used to compare three kits for the detection of H pylori antibodies: (1) Pyloriset Dry, total antibody latex agglutination, Orion Diagnostica, Espoo, Finland; (2) Pyloriset enzyme immunoassay (EIA), IgG ELISA, Orion; and (3) Hel-p, IgG ELISA, Amrad, Kew, Victoria, Australia. Diagnosis of H pylori positivity was made if culture results and either rapid urease test or histopathology were positive. The sensitivity, specificity, positive, and negative predictive value for each test was as follows: Orion: latex 93.3%, 95.6%, 95.5%, 93.3%, respectively; Orion: EIA-G 84.4%, 97.8%, 97.4%, 84.4%, respectively; and Amrad: EIA-G 100%, 88.9%, 90%, 100%, respectively. The latex test performed better than the EIAs with respect to sensitivity and specificity.

Random amplified polymorphic DNA and plasmid analyses used in investigation of an outbreak of multiresistant Klebsiella pneumoniae.

Multiresistant Klebsiella pneumoniae strains with plasmid-borne extended-spectrum beta-lactamases (ESBL) are increasingly frequent nosocomial pathogens. A major outbreak of clinical infections, mainly involving patients in the Newborn Services Unit with limited spread to adult patients, occurred at our hospital. This epidemic was investigated by typing the isolates phenotypically and with random amplified polymorphic DNA analysis (RAPD) and plasmid analysis. Forty-eight isolates, consisting of 44 consecutive clinical isolates and 4 selected surveillance isolates, were studied. A single decamer primer was used for the RAPD, and this was effective in demonstrating that the majority of isolates (45 of 48) had the same profile. Three other isolates had different RAPD patterns identifying them as nonepidemic strains. Plasmids were extracted by alkaline lysis with Magic-miniprep kits from 10 isolates selected to represent the epidemic and nonepidemic strains. This method produced small (< 20-kb) plasmids; larger ESBL-carrying plasmids were not produced, but the small plasmids nonetheless allowed strain differentiation. Antibiotic susceptibility patterns alone were not reliable as strain indicators, since some isolates with the RAPD pattern characteristic of the epidemic strains did not express ESBL and therefore were susceptible to extended-spectrum cephalosporins. The investigation showed the predominance of a single epidemic strain that was transmitted between patients in the Newborn Services Unit. RAPD was the best of the methods used for detecting strain differences, and its speed and ability to type a wide variety of species suggest that it will be an increasingly useful molecular epidemiologic tool.

Comparison of immunofluorescent antibody test and enzyme-linked immunosorbent assay for detection of antibodies to Listeria monocytogenes.

This laboratory has used an immunofluorescent antibody test (IFAT) for the detection of IgG and IgM antibodies to Listeria monocytogenes. This test is performed using whole cells of serotypes 1/2a, 1/2b, and 4b as antigen. Sera are pre-absorbed to remove cross-reacting antibodies. This test can diagnose listeriosis by demonstration of seroconversion in appropriately spaced acute and convalescent sera. The disadvantages of this test are the necessity for pre-absorption, the use of three different test antigens and the need for subjective interpretation. An enzyme-linked immunosorbent assay (ELISA) using sonicated listeria cells as the antigen in a microtitre plate system can theoretically overcome these shortcomings. An ELISA for antilisterial IgG and IgM was compared to the IFAT using sera from bacteriologically confirmed cases as positive controls and sera submitted for routine rubella serology as negative controls. The ELISA method as tested appeared to be less specific than the IFAT. As the serological diagnosis of listeriosis is complicated by the failure of the main antibody response to switch from IgM to IgG after infection, asimilar ELISA system was used to detect anti-listerial IgA antibody. This assay detected IgA in patients with proven listeriosis but not in the control patients. It appears to be sensitive, specific and easy to perform.

Isolation of mycobacteria inducing cross-reactions in the complement fixation test for Johne's disease.

Eight bulls which gave positive reactions in the complement fixation test for Johne's disease were identified in a group of 29 bulls which had previously given only negative reactions at repeated annual tests. One reacting bull was confirmed to be infected with Mycobacterium paratuberculosis at autopsy, but another reactor which was slaughtered had no visible lesions and mycobacteria were not recovered in culture. Faecal samples from seven further bulls (six reactors and one non-reactor) yielded mycobacteria which were not dependent on mycobactin for growth. When one of these strains was injected into two calves they developed positive reactions in the complement fixation test for Johne's disease and to intradermal skin testing with purified protein derivatives of M avium, M paratuberculosis and M bovis. The strains did not satisfy the exact requirements for classification into any recognised mycobacterial species.

Streptobacillary pleuritis in a koala (Phascolarctos cinereus).

A case of Streptobacillus moniliformis pleuritis in a koala (Phascolarctos cinereus) is reported. Lesions were granulomatous in nature. S. moniliformis was recovered in pure culture, and found by experimental inoculation to be pathogenic for mice but not for a rat.

Mycobacterium intracellulare infection in a water monitor.

Mycobacteriosis caused by Mycobacterium intracellulare serotypes Davis (8) and Altman (18) is described in a water monitor (Varanus semiremex). Infection with this of organism has not been reported previously in reptiles in Australia.

Types and distribution of anaerobic bacteria in the large intestine of pigs.

An examination was made of various sites along the length of the swine large intestine, using strictly anaerobic culture methods. Sites were separated by differential washing into fractions described as lumenal content, lumenal surface layer, and intestinal wall tissue. Direct microscopic clump counts averaged 13.3 x 10 organisms per g (dry weight) of material in the lumenal content, 14.0 x 10 in the surface layer, and 5.1 x 10 in the intestinal wall tissue. Both direct microscopic counts and viable culture counts were higher from the lumenal content and surface layer than from the intestinal tissue at all sites sampled in the intestine. Cultural counts averaged 56.2% of the direct microscopic counts in lumenal content and surface layer and 20.2% in intestinal tissue. Over 90% of the bacteria isolated were gram positive and consisted mainly of gram-positive cocci, lactobacilli, eubacteria, and clostridia. Of 192 isolates recovered, only 124 could be assigned to recognized species.

Haemophilus parasuis infection in swine.

Septicemic disease occurred in 49 of 126 pigs several days after being transported 80 km. All affected pigs died. The main changes in acutely affected pigs were skin discoloration, pulmonary edema, arthritis, meningitis, and renal glomerular thrombosis. In peracute cases, gross findings were minimal. Haemophilus parasuis was isolated from multiple organ sites in most affected pigs. Haemophilus parasuis was isolated from nasal swab specimens from 17 of 20 clinically normal pigs on the farm of origin. Fatal acute septicemia was reproduced in 2 pigs by intravenous or intratracheal exposure to an isolant of H parasuis obtained from 1 of of the 49 fatally affected pigs. Aerosol exposure of 5 pigs resulted in mild pneumonia in 4 pigs and severe pneumonia, pleurisy, pericarditis, and terminal septicemia in 1 pig.