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Although methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (RVS-MRSA; including vancomycin-intermediate S. aureus [VISA] and heterogeneous VISA [hVISA]) have been linked with vancomycin treatment failure, it is unclear whether they are more pathogenic than vancomycin-susceptible MRSA (VS-MRSA). We prospectively assessed patients with clinical MRSA isolates during a 10-month period to determine clinical status (infection versus colonization) and therapeutic outcome before correlating these findings with the results of detailed in vitro assessment of vancomycin susceptibility, including population analysis profile (PAP) testing. hVISA and VISA were defined by standard PAP criteria (area-under-the-curve ratio compared to that of the reference hVISA strain Mu3 [>or=0.9]) and routine CLSI criteria (vancomycin MIC, 4 to 8 microg/ml), respectively. Among the 117 patients assessed, 58 had RVS-MRSA isolates (56 hVISA and 2 VISA) and 59 had VS-MRSA isolates; the patient demographics and comorbidities were similar. RVS-MRSA was associated with a lower rate of infection than VS-MRSA (29/58 versus 46/59; P = 0.003), including a lower rate of bacteremia (3/58 versus 20/59, respectively; P < 0.001). The cure rates in RVS-MRSA and VS-MRSA groups were not statistically different (16/26 versus 31/42; P = 0.43), but the post hoc assessment of treatment regimes and study size made detailed conclusions difficult. The results of the macro method Etest correlated well with the PAP results (sensitivity, 98.3%, and specificity, 91.5%), but broth microdilution and our preliminary RVS-MRSA detection method correlated poorly. All isolates were susceptible to linezolid and daptomycin. These data suggest that detailed prospective laboratory identification of RVS-MRSA isolates may be of limited value and that, instead, such in vitro investigation should be reserved for isolates from patients who are failing appropriate anti-MRSA therapy.

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To develop a double gradient denaturing gradient gel electrophoresis (DG-DGGE) based typing method that rapidly and accurately types clinical isolates of Staphylococcus aureus, the VS2 region of the 16S-23S rRNA spacer region (ISR) was chosen because of its potential high variation. The VS2 region was amplified with a 40-mer GC-clamp attached to the 5'-end of the reverse primer. The 145 bp PCR product was then separated by DG-DGGE using denaturant concentrations of 25-40% and polyacrylamide concentrations of 6-12%. Of the five mutations identified in 336 S. aureus isolates, one mutation was found to be highly specific for 161/171 (94%) of methicillin-resistant S. aureus (MRSA) isolates from different geographic locations and isolation times. This same mutation was found in 15/160 (9%) of penicillin- or methicillin-sensitive S. aureus isolates. In some isolates two mutations occured together in the one genome suggesting some S. aureus isolates have two copies of VS2. In these 336 isolates nine genotypes with different combinations of the five mutations were identified. In 18 coagulase-negative staphylococci (CNS), the MRSA-specific mutation was found along with two other mutations in all isolates demonstrating consistent differences in the presence of these mutations between CNS and S. aureus. The marked differences in VS2 sequences found between MRSA, methicillin- or penicillin-sensitive S. aureus (SSA), and CNS by DGGE in the present study may be useful in evolutionary studies and in the development of a specific assay for MRSA from clinical specimens.

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A nocardioform bacterium was isolated from the bronchoscopic lavage of a 78-year-old man with a past history of tuberculous pleurisy, who presented with bilateral upper lobe lesions at Austin and Repatriation Medical Centre, Heidelberg, Australia. The strain was aerobic, Gram-positive, produced beige substrate mycelium and scant white aerial mycelium. It showed chemotaxonomic markers which were consistent with the classification of Nocardia: i.e. meso-diaminopimelic acid, N-glycolylmuramic acid, arabinose and galactose as diagnostic sugars; phospholipids phosphatidylinositol mannosides, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol; a menaquinone with a cyclic isoprene side chain, MK-8(H4cycl.); a fatty acid pattern composed of unbranched saturated and monounsaturated fatty acids with a considerable amount of tuberculostearic acid; and mycolic acids composed of 54-62 carbon atoms with three principal mycolic acids which were mono- and polyunsaturated, showing a chain length C56, C58 and C60 and accounting for over 70% of the entire pattern. The 16S rDNA sequence showed the highest similarity to the type strain of Nocardia vaccinii; the DNA-DNA similarity of the two strains was 31%. These data, together with distinct physiological traits and molecular biological analyses, as well as chemotaxonomic results, led to the conclusion that the novel isolate represents a new species within the genus Nocardia for which the name Nocardia veterana sp. nov. is proposed. The type strain is M157222T (DSM 44445T = NRRL B-24136T).

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The current literature on bacterial taxonomy, typing and evolution will be critically examined from the perspective of whole-genome structure, function and organization. The following three categories of DNA band pattern studies will be reviewed: (i) random whole-genome analysis; (ii) specific gene variation and (iii) mobile genetic elements. (i) The use of RAPD, PFGE and AFLP to analyse the whole genome will provide a skeleton of polymorphic sites with exact genomic positions as whole-genome sequence data become available. (ii) Different genes provide different levels of evolutionary information for determining isolate relatedness depending on whether they are highly variable (prone to recombination events and horizontal transfer), housekeeping genes with only a small number of single nucleotide differences between isolates or part of the rrn multigene family that is prone to intragenomic recombination and concerted evolution. Comparative analyses of these different gene classes can provide enhanced information about isolate relatedness. (iii) Mobile genetic elements such as insertion sequences, transposons, plasmids and bacteriophages integrate into the bacterial genome at specific (e.g. tRNA genes) or non-specific sites to alter band patterns produced by PFGE, RAPD or AFLP. From the literature it is not clear what level of genetic element duplication constitutes non-relatedness of isolates. A model is presented that incorporates all of the above genomic characteristics for the determination of isolate relatedness in taxonomic, typing and evolutionary studies.

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We report the first instance in Australia of treatment failure due to a strain of methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin--heteroresistant vancomycin-intermediate S. aureus (hVISA). The infection occurred in a 41-year-old man with multiple risk factors. No transmission of the organism to other patients or the environment was detected. This case may herald the beginning of a new phase of staphylococcal resistance in Australia.

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Proliferation indices are intended to help patients and clinicians make treatment decisions. We have previously demonstrated that a proliferation index based on in vivo labeling of S-phase cells with bromodeoxyuridine (BrdUrd) correlates with Ki-67 labeling index (LI). We now compare the prognostic value of these indices. With written consent, we gave 129 women with biopsy confirmed breast cancer 200 mg/M2 BrdUrd during 30 min immediately preceding surgery. We used IU-4 anti BrdUrd antibody to count the immunohistochemical labeling index (LI) of DNA-incorporated BrdUrd in 2,000 cells and MIB-1 to count Ki-67 (118 cases). Patients received standard surgical and adjuvant treatment. No patients were lost to follow-up and patients were followed a minimum of 2 (median 5.1) years. We compared survival and recurrence in tumors with high vs low labeling indices. We found that women in the low BrdUrd LI group had better disease free survival (92% vs 67% 5-yr DFS p = 0.001) and overall survival (94% vs 70% 5-yr OS, p = 0.0001) than those with a high LI. In comparison, a low Ki-67 index predicted better OS (87% vs 80% 5-yr OS, p = 0.020) and a trend for better DFS (84% vs 72% DFS p = 0.055). The apparent superiority of BrdUrd LI over Ki-67 LI is likely due to chance (p = 0.18). In multivariate survival analyses we found that BrdUrd LI proliferative index significantly improves prediction of DFS or OS even when node status, age or tumor size is in the model. We conclude that markers of proliferation are useful adjuncts in predicting patient prognosis.

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Gilbert's syndrome, due to reduced hepatic bilirubin glucuronidation is associated with the presence of two extra nucleotides (TA) in the promoter region of the UDP-glucuronosyltransferase 1 (UGT1A1) gene. A rapid method was developed to detect this genetic polymorphism, using double gradient denaturing gradient gel electrophoresis (DG-DGGE). The promoter region of the UGT1A1 gene was amplified with a 40-mer GC-clamp attached to the 5'-end of the reverse primer. The polymerase chain reaction (PCR) product was then separated by DG-DGGE using denaturant concentrations of 15-25% and polyacrylamide concentrations of 6-12%. The (TA)6/(TA)6 homozygotes were clearly distinguished from both (TA)7/(TA)7 homozygotes and (TA)6/(TA)7 heterozygotes. The (TA)7 allele frequency was consistent with that previously reported and elevated bilirubin levels correlated with the presence of the (TA)7 allele. The DG-DGGE method described will make detection for this polymorphism fast, simple, nonradioactive and suitable for a clinical routine diagnostic laboratory, helping to establish the role of this polymorphism in individuals with jaundice due to multiple causes.

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A 53-year-old woman from Melbourne, Australia, with squamous cell carcinoma of the oesophagus was shown by computed tomography (CT) scan to have a left apical cavity and inflammatory changes in the right lung consistent with aspiration. Acid-fast bacilli isolated from bronchial washings were identified biochemically first as Mycobacterium terrae, but later as M. shimoidei on the basis of 1) restriction fragment analysis and 2) sequencing of polymerase chain reaction (PCR) amplified 16S rDNA. Nine other descriptions of patients with M. shimoidei isolates were collated. The salient feature of isolates considered to be pathogenic was pulmonary cavitation. Most patients had underlying lung disease, including past tuberculosis or malignancy. Six of eight patients died of progressive respiratory illness, although the contribution of M. shimoidei was not always clear, and two patients improved. One patient with the acquired immune-deficiency syndrome (AIDS) died with Salmonella enteritidis and M. shimoidei isolated from blood cultures. One isolate was regarded as a coloniser. There are insufficient clinical or sensitivity data on which to base recommendations for therapy, but a combination of ethambutol, rifabutin and pyrazinamide could be considered.

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We report the isolation of Clostridium tetani (along with Fusobacterium mortiferum) from empyema pus. The patient, a 68 year old retired farmer from rural NSW, had recently undergone cholecystectomy, had heart failure and developed an empyema. He improved after drainage of the empyema and penicillin therapy, but died suddenly during convalescence.

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A case of Mycobacterium shimoidei in a 75 year old man is reported. He had been a smoker, with a past history of bullous emphysema and a lung abscess. He had a 12 month history of weight loss, night sweats, with increased cough and sputum, and progressive opacification of the left apex with cavity formation. Sputum repeatedly grew M. shimoidei, identification of which was confirmed with high-pressure liquid chromatography (HPLC). He was treated for 45 days with three drugs to which the organism was sensitive, but failed to respond. His death was attributed to mycobacterial infection.

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Proliferation indices are used, along with other parameters, to estimate the risk of recurrence of breast cancer for individual patients. Because it is unlikely one index will be practical for all patients, it is important to understand the relationship between various indices of proliferation. For this reason, we compared a proliferation index based on in vivo labeling of S-phase tumor cells with the thymidine analog bromodeoxyuridine (BrdUrd), to a proliferation index based on an estimate of the growth fraction with the MIB-1 antibody to the Ki-67 antigen. With informed consent, we gave 145 patients 200 mg/m2 BrdUrd intravenously just prior to surgical removal of breast cancer. On histology sections, we visually counted S-phase cells which had incorporated BrdUrd using the Br-3 antibody which is specific to DNA-incorporated BrdUrd, and we counted cells in the growth fraction using the MIB-1 antibody to the Ki-67 antigen. We found that both indices were positively correlated with tumor size, number of positive nodes, and tumor grade, and both were negatively correlated with age and estrogen-progesterone receptor positivity. Using a linear functional relationship model, we found that the best (i.e. the maximal) fit between the two indices (correlation coefficient 0.79; p < 0.0001) occurred when each index was square root transformed, as is appropriate when counts follow a Poisson distribution. When we used the median as a cutpoint for each index, the classification of 19 percent of data pairs changed depending upon which index was used. We also estimated that the Ki-67 intercept (1.02 +/- 0.25) was significantly greater than zero. We conclude that the BrdUrd index of DNA synthesis in S-phase correlates highly with the MIB-1 index of the growth fraction, and both indices correlate well with other parameters of tumor aggressiveness. Because this correlation is driven by concordance of the extremes of high and low counts, clinical comparison will be necessary to determine which is the better prognostic marker for human breast cancer.

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Most research on children and risk is heavily influenced by developmental theory. This paper is based on a study which uses a different approach, drawing on recent work within the sociology of childhood. 'Children, Parents and Risk' explores the ways in which risks to children are understood and managed by children and parents, focusing on children's daily lives in and around the home at the ages of 3, 9 and 12 years. Data were gathered from interviews with children and their parents at home and from children at school and in a youth club. The paper draws on the findings from the study in order to discuss and compare parents' and children's ideas about children and childhood as risk-related; it also aims to examine the findings in the context of Ulrich Beck's recent work on the risk society and individualization. Both parents and children tended to 'externalize' risk away from the home and into the outside world. In response to perceived risks to childhood many parents appeared to see their role as that of striking a balance between protection and compensatory provision, and their accounts included details of increasingly 'individualized' measures to reduce such risks. The children conceptualized their experiences of adult control as welcoming when preventing the child's exposure to risk but as constraining when it restricted their autonomy. The paper concludes that the findings accord with Beck's description of the 'risk society' and that they lend some limited support to Beck's individualization thesis.

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The antibacterial activity of meropenem was tested against 426 clinical isolates representing a wide range of aerobic and anaerobic species. The in vitro activity of meropenem was compared with that of iminpenem, ceftazidime, cefotaxime, ciprofloxacin, piperacillin and tobramycin against aerobic isolates, and also compared with that of imipenem, metronidazole, cefoxitin, clindamycin and piperacillin against the anaerobic isolates. Meropenem exhibited an extended spectrum of activity with low minimal inhibitory concentrations (MIC) against Gram negative aerobes, anaerobes, Gram positive anaerobes and most of the Gram positive aerobes. The MIC90 of meropenem against the Enterobacteriaceae ranged from 0.03 mg/l to 0.125 mg/l. Meropenem was very active against extended spectrum beta lactamase producing Klebsiella pneumoniae, most Acinetobacter species, Pseudomonas aeruginosa, Clostridium difficile (MIC90 of 1.0 mg/l), Clostridium perfringens and other Clostridium species. Even though imipenem exhibited better activity against the coagulase negative staphylococci, meropenem still had MIC's which were less than the break point (8.0 mg/l). The stability of meropenem in agar was determined indirectly by plotting the geometric mean MIC of control strains over a period of two weeks. Mean MIC of six control strains for meropenem was 0.05 mg/l and remained constant over 10 days, whereas the mean MIC of imipenem rose to 0.24 mg/l after two days and to 3.4 mg/l after 10 days. Meropenem was therefore far more stable in agar than imipenem. This has implications for laboratories using agar dilution as it requires less frequent plate preparation and decreased labour costs.

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In December 1992, a thoracic ward in a Melbourne teaching hospital experienced an increase in patients infected with methicillin-resistant Staphylococcus aureus (MRSA). It was decided to attempt to control the outbreak by cohorting positive patients (infected and colonized), as well as nurse cohorting, emphasis on handwashing, and use of intranasal mupirocin initially three times a day for three days, then thrice weekly, for all patients in the ward (with or without MRSA). The campaign comprised for phases of 53, 45, 92 and 365 days, respectively. Patient and nurse cohorting stopped at the end of phase I. In phases I and II, surveillance nose swabs were taken on admission, then twice weekly; in phase III, on admission and weekly and in phase IV, on admission until the end of 1993. In phases I and II (98 days), only one patient acquired MRSA. When the frequency of mupirocin prophylaxis was decreased to once weekly (phase III), two patients acquired MRSA in 92 days (no significant difference): thrice weekly administration resumed (phase IV), during which there were three acquisitions in 365 days. The rates of nose colonization of admissions were 6.4%, 6.3%, 9.7% and 3.1% in phase I-IV, respectively. Only three patients were treated with vancomycin between July 1993 and June 1994 (significantly lower than historical rates, P = 0.0086). No mupirocin resistance was seen in MRSA isolates from this ward during phases I, II and III. In areas of low-level endemic MRSA, the blanket use of thrice-weekly intranasal mupirocin may be effective in decreasing serious infections with MRSA, and does not necessarily elicit mupirocin resistance.

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BACKGROUND: Tumor angiogenesis, as assayed by microvessel density, has been proposed as an independent prognostic marker for clinical outcome in breast cancer patients. PURPOSE: The present study evaluated the microvessel density assay and assessed its utility, alone and together with the evaluation of other tumor characteristics, in predicting outcome in patients with invasive ductal carcinomas. METHODS: In a blinded design, cases of invasive ductal carcinoma were selected from a registry containing the records of and tumor specimens from 386 breast cancer patients treated at the Massachusetts General Hospital from 1977 through 1982. After the exclusion of ineligible patients and inadequate specimens, 220 patients were included in the study; their median time of follow-up was 11.5 years. Half of these patients (n = 110) were positive for axillary lymph node metastases. Histologic sections of the tumors were stained immunocytochemically for factor VIII, a coagulation protein expressed by blood vessel endothelium, and for p53 protein. Independently, two analysts counted microvessels in three microscope fields selected from separate vascular regions of the tumor. Variability in microvessel scores between analysts and among different fields of the same tumor was summarized by the coefficient of variation. The kappa statistic tested for agreement between the analysts while correcting for chance agreement. The effects of tumor characteristics on metastasis-free survival and overall survival were tested univariately by the Harrington-Fleming rank test procedure. The effect of multiple factors on survival was tested under a Cox multivariate proportional hazards model. RESULTS: Microvessel count showed considerable variability between the two analysts and among regions within each tumor, with an overall concordance for tumor classification of 73%. Univariate analysis revealed no association between microvessel count and any other tumor or patient characteristic. Multivariate analysis indicated, for these patients, that nodal status and p53 staining predicted metastasis-free survival and that nodal status, estrogen receptor (ER) status and tumor grade predicted overall survival. CONCLUSIONS: Microvessel count showed much variation among different regions of each tumor. It did not predict metastasis-free survival or overall survival. Nodal status was the most powerful criterion to stratify these patients with invasive ductal carcinoma of the breast into different survival groups. Only ER status, tumor grade, and p53 staining had additional prognostic utility for these patients after they had been stratified by nodal status.

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The process of selection of a pathology computer system has become increasingly complex as there are an increasing number of facilities that must be provided and stringent performance requirements under heavy computing loads from both human users and machine inputs. Furthermore, the continuing advances in software and hardware technology provide more options and innovative new ways of tackling problems. These factors taken together pose a difficult and complex set of decisions and choices for the system analyst and designer. The selection process followed by the Microbiology Department at Heidelberg Repatriation Hospital included examination of existing systems, development of a functional specification followed by a formal tender process. The successful tenderer was then selected using predefined evaluation criteria. The successful tenderer was a software development company that developed and supplied a system based on a distributed network using a SUN computer as the main processor. The software was written using Informix running on the UNIX operating system. This represents one of the first microbiology systems developed using a commercial relational database and fourth generation language. The advantages of this approach are discussed.

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Abnormal accumulation of the p53 tumor suppressor gene product was analyzed in 198 primary invasive human breast carcinomas. In 47 of these cases, single-strand conformational polymorphism was used to detect mutations in the highly conserved exons 5-9 of the gene. Mutations as determined by single-strand conformational polymorphism were found in 15 of 15 strongly immunopositive cases (100%) and 3 of 23 immunonegative cases (13%). There were also nine cases with < 1% immunopositive cells (borderline immunopositivity); p53 mutations were detected in seven of these cases. The results suggest that p53 immunopositivity is a highly specific, albeit somewhat insensitive surrogate for p53 mutations. p53 accumulation, detected by immunohistochemical methods using antibody PAb 1801, was noted in 29.8% of the cases and was associated with estrogen receptor (ER) negativity (P = 0.0003), progesterone receptor (PR) negativity (P = 0.008), and high histological grade (P = 0.037) by univariate analysis. Incorporation of bromodeoxyuridine was used to determine the percentage of cells synthesizing DNA (proliferative fraction). When bromodeoxyuridine was administered either in vivo (n = 93) or in vitro (n = 79), p53 accumulation was only marginally related to proliferative fraction (P = 0.067 by chi 2; P = 0.055 by Mann-Whitney). When tumors were segregated by ER status, the aforementioned associations of p53 immunopositivity with PR negativity, high histological grade, and increased proliferation rate lost their significance. p53 accumulation did not correlate with tumor size, clinical stage, axillary node metastases, or age at diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

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