A novel selective medium for the detection of methicillin-resistant Staphylococcus aureus enabling result reporting in under 24 h.

A simple selective broth was devised to indicate the presence of methicillin resistant Staphylococcus aureus (MRSA) in clinical samples. The broth comprised nutrient broth supplemented with sodium chloride, ciprofloxacin, colistin and aztreonam as selective agents and also mannitol, trehalose and phenol red as an indicator system. In a preliminary study using 228 clinical samples this selective mannitol broth (SMB) proved to be more sensitive than other selective agars for detection of MRSA within 24 h. In an extended study of a further 1124 clinical samples from 470 randomly selected patients, SMB detected 85.1% of MRSA strains present with a specificity of 43.6%. We conclude that SMB offers a convenient, inexpensive and sensitive method for high-throughput screening for MRSA.

Survival of enterococci during hospital laundry processing.

Much remains to be elucidated about the epidemiology of nosocomial enterococcal infections. Enterococci are, however, known to be relatively thermotolerant, and several studies have shown that under laboratory conditions many strains are able to survive the time/temperature combinations of the UK Department of Health recommendations for the decontamination of used linen (HSG(95)18). We therefore wished to investigate the efficacy of decontamination of enterococci from hospital linen in working hospital laundries. The thermotolerance of 40 strains of Enterococcus faecalis and Enterococcus faecium was first determined. Reduction by a factor of greater than 10(5) was achieved in only two of 40 strains after 3 min at 71 degrees C or 10 min at 65 degrees C, the time/temperature combinations specified by the Department of Health for the disinfection of used linen. During experimental challenge of 10 working hospital laundries, however, we demonstrated successful decontamination of laundry artificially contaminated with enterococci. This was shown to take place during the washing stage. Our study suggests that, despite the relative thermotolerance of enterococci, the time/temperature combinations specified in HSG(95)18 should be adequate for their decontamination in hospital laundries.

Heat disinfection of clinical waste: microbiological assessment and monitoring of effectiveness.

Simple, rapid and reproducible protocols are described for the microbiological assessment of clinical waste treatment processes, using Bacillus subtilis spore tests and end-product sampling. The use of these protocols to commission a new heat disinfection system (HDS), based on a hot oil-filled auger, and to monitor it over the first 21 months of operation is described. It is suggested that these protocols are suitable for assessment of other non-burn heat-treatment technologies.

Rapid identification of Staphylococcus aureus by using fluorescent staphylocoagulase assays.

Two rapid (1-h) assays for the detection of Staphylococcus aureus staphylocoagulase were developed by using the fluorogenic thrombin substrates N-t-boc-Val-Pro-Arg-7-amido-4-methylcoumarin (VPA) and N-t-boc-beta-benzyl-Asp-Pro-Arg-7-amido-4-methylocoumarin (BB). The assays were compared to the tube coagulase test and latex agglutination (LA) (Sanofi Diagnostics Pasteur, Guildford, Surrey, United Kingdom) by using 406 clinical isolates of staphylococci, and they produced positive and negative predictive values of 99.2 and 99. 1% for LA, 98.9 and 92.7% for VPA, and 98.9 and 99.1% for BB. Fluorescent assays used colonies from solid media, thereby eliminating the need for broth cultures, and were performed in microtiter trays, thus making them suitable for large-scale screening.

Evaluation of tube coagulase and a fluorogenic substrate for rapid detection of methicillin-resistant Staphylococcus aureus from selective enrichment broth in an outbreak of EMRSA 15.

We investigated the use of tube coagulase and a fluorescent substrate, N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin for the rapid detection of MRSA in selective broth enrichment cultures during an outbreak. These methods were compared with direct plating of swabs and plating a selective broth enrichment culture using 200 screening swabs collected from forty patients during the investigation of an outbreak of E-MRSA 15. Overall 66 swabs were positive for MRSA following subculture of broth enrichment culture. Direct plating detected 25 (38%) positives, tube coagulase 37 (56%), and fluorescent substrate 49 (74%) respectively, although nine of the 49 turned out to be false reactions. When detection from individual patients was analyzed, selective broth subculture identified 28 patients colonized with MRSA. Direct plating detected only 12 (43%) of these patients. The tube coagulase and fluorescence methods detected MRSA in 17 (60%) and 19 (68%) patients respectively. The tube coagulase method was found to be 100% specific for MRSA suggesting its use as a rapid method for the detection of MRSA from selective enrichment broth.

Use of latex agglutination technique for detecting Legionella pneumophila (serogroup 1) antibodies.

Following the outbreak of Legionnaires' disease in Stafford in 1985, 500 serum samples were submitted to the indirect immunofluorescence antibody test and a latex agglutination. Latex agglutination using ultrasonically disrupted Legionella pneumophila antigens coupled to latex particles, proved a rapid, simple method for detecting circulating antibodies to L pneumophila in a one minute slide latex agglutination test. There was good correlation with the indirect immunofluorescence antibody test (IFAT), and the specificity and sensitivity with respect to a diagnostic result were 98.3% and 97.6%, respectively, using a series of well characterised sera. The latex agglutination test seems well suited as a screening test for presumptive cases of Legionnaires' disease; the latex reagent is easy to prepare and seems to remain stable at 4 degrees C for up to six months.

Pneumococcal typing by polyvalent counterimmunoelectrophoresis (PIE).

A one-step polyvalent counterimmunoelectrophoresis (PIE) method for the typing of pneumococci is describe. Only one antiserum is used (omniserum, containing antibodies to greater than 80 pneumococcal types) and it is not necessary to stock large numbers of monospecific typing sera. By observing reactions of identity between the precipitin lines produced by the pneumococcus under test, and the specific capsular polysaccharide precipitin lines in a reference pattern produced by the polyvalent pneumococcal vaccine 'Pneumovax', a cumulative percentage of 64.6% of pneumococcal types isolated could be typed. The method is simple, reproducible, inexpensive and provides a permanent stained record.

The diagnosis of Legionnaires' disease by counterimmunoelectrophoresis.

Counterimmunoelectrophoresis (CIE) was found to be a rapid, specific method for detecting circulating antibodies to Legionella pneumophila, the aetiologic agent of Legionnaires' disease in human sera. Optimum conditions for performing the test are given. Better precipitin lines are observed when the gel support is neutral, and a result may be obtained in 90 to 180 minutes. Comparison of results of CIE and indirect fluorescent antibody tests on 22 sera from patients with Legionnaires' disease and on 27 sera from healthy control subjects showed 100% correlation, and 75 paired sera from patients with pneumonia of unknown aetiology showed 96.7% correlation. Laboratory diagnosis may be made quickly and accurately by CIE. The method is simple and easily performed in the routine laboratory. The antigen is stable at 4 degrees C for at least six months.

A modified cytoplasmic antigen of Candida albicans for serodiagnosis of systemic candidiasis.

A modified cytoplasmic antigen, prepared by Natamycin degradation of Candida albicans cells is described. The reactivity of this antigen in detecting precipitins to Candida albicans proved tobe very similar qualitatively and quantitatively to a standardised reference antigen, prepared by X-press disruption when both were compared by immunological techniques; the majority of antigenic components in each proving to be identical. When tested against 127 human sera of unknown antibody content the two antigens showed 100% correlation by counterimmunoelectrophoresis and 87.5% correlation by double diffusion. The modified antigen proved to be reproducible and reliable in use and is easily prepared in the routine laboratory.