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Rapid detection and molecular characterization of australian human rickettsial isolates
40 ENDOTOXIN AS A MARKER OF GRAM NEGATIVE BACTERAEMIA IN FEBRILE NEUTROPAENIC PATIENTS J.C. Hurley & J.H. Andrew, Microbiology Department, StVincent's Hospital, Melbourne. Endotoxin is a cell wall component of Gram negative bacteria. Patients with neutropaenia, such as patients with haematological matignancies, are at greatly increased risk of infection with these bacteria. An assay for endotoxin would assist in the early detection of Gram negative infections and also, in monitoring the response of these patients to antibiotic therapy. However, endotoxin is unstable in plasma. We have developed the optimal method for pretreatment of plasma (dilution & heating) and the appropriate assay methodology (kinetic,turbiimetric) using the Limulus Amoebocyte lysate reagent (Hurley et a1.1988, Abst #.I55 Aust. Micr. 9(2) 216). Blood samples were obtained from patients who were receiving antibiotic therapy during periods of neutropaenic sepsis. The samples were obtained prior to and at approximateiy 2 hours following the administration of the morning dose of antibiotic. The endotoxin levels were higher in the post antibiotic samples than the preantibiotic samples only for the subgroup of patients who had blood culture documented Gram negative sepsis. The magnitude of the increase was similar to that observed previously in a urinary tract model (Hurley et a1.1989, Abst #.26.6Aust. Micr. 1Q (3)383).
Presenter; Dr James Hurley, Microbiology Department, SV H
ABSTRACTS
RAPID DETECTION AND MOLECULAR CHARACTERIZATION OF AUSTRALIAN HUMAN RICKE'ITSIAL ISOLATES. Baird R W*, Ross B, Stenos J, Dwyer B. Clinical Pathology Laboratories, Fairfield Hospital, Fairfield, Victoria, 3078. Australia has a number of unique rickettsial diseases including Queensland Tick Typhus. More recently, Flinders Island Spotted Fever ( FISF ) of presumed rickettsial origin has been described. We have studied Australian human rickettsial disease from 2 perspectives. Firstly, we have detected rickettsial DNA in blood samples of 2 patients with FISF by DNA amplificationutilizing the Polymerase Chain Reaction. Confirmation of the rickettsial origin of the amplified DNA was made by hybridizationwith a rickettsiaspecific DNA probe. This probe, made from the genus-common 17 kilodalton antigen of Rickettsia australis has been fully characterised and incorporates non-radioactive detection of target molecules. We aim to develop this method to provide rapid diagnosis of rickettsia1 disease Secondly, by applying the molecular typing technique of ribosomal DNA hybridization we have investigated the DNA relatedness of Australian human rickettsial isolates from Queensland Tick Typhus and presumptive human rickettsial isolates from RSF. Digestion of purified rickettsial chromosomal DNA from human isolates with the restriction endonuclease Eco R1 revealed specific differences between rickettsial isolates from patients with Queensland Tick Typhus and FISF when probed with ribosomal DNA. Ongoing studies are in progress to establish the relatedness of these isolates. Presenter: Dr. Robert Baird, Fairfield Hospital
CULTURE POSITIVE TUBERCULOSIS: A 28 YEAR REVIEW. J.C. Hurley, E.M. Uren & J.H. Andrew, Microbiology Department, SLVincent's Hospital, Melbourne. Between 1962 and 1989, 36,000 specimens were submitted to the microbiology laboratory for examination for Mycobactefium tuberculosis (MTB). The annual number of specimens has increased from around 1,000 to 1,900 per annum over this period. We reviewed the types of specimens submitted and found a greater yield from gastric aspirate and tissue specimens in comparison to other specimens. Demographic information on the patients that were found to be culture positive for MTB was obtained from SVH medical records and notification records (through the Tuberculosis program, Health Department, Victoria). An increase in the numbers of patients whose country of birth was in Asia or the Mediterranean area was noted and these patients accounted for the majority of tuberculosis in the 1980's. The patients from these countries present at a younger age, with a higher proportion of extra-pulmonary disease and a higher proportion of drug resistant disease than patients born in Australia or Europe. Presenter; Dr James Hurley, Microbiology Department, SV H
PENICILLIN TOLERANCE IN VWDANS STREPTOCOCCI CAUSING ENDOCARDITIS - A NOVEL DETECTION METHOD EMPLOYING THE "E-TEST"
R. Bunter, K. Stockman, J. Downes, J. H. Andrew, Department of Microbiology, St. Vincent's Hospital, Melbourne, Vic. 3065. Viridans streptococci (VS) causing bacterial endocarditis are frequently tolerant to penicillin (MIC:MBC ratio 232) and a treatment combination of a penicillin and an aminoglycoside is commonly used. If the absence of tolerance could be simply and rapidly determined, there is an option of ceasing or shortening the duration of aminoglycoside therapy in patients where toxicity from this group of drugs is of concern. Determination of the MIC and MBC of VS causing endocarditis has traditionally required labour intensive broth dilution methods. MBC estimations have been poorly standardised are subject to technical error and available in few laboratories. A new method, the "E-Test" (AB Biodisk) allows the simple and accurate determination of MIC's. A modification of this method to enable the determination of tolerance was developed. Following estimation of the MIC residual penicillin was inactivated with Beta-lactamase delivered by a measured dose spray, the plate reincubated and surviving organisms in a specified area counted. Forty VS isolates from cases of proven endocarditis were examined for tolerance using the "E-Test" and traditional micro and macro broth dilution methods (NCCSLS M7-T2 and M26-P). The "E-Test" method compared very favourably, was relatively non-labour intensive, less subject to technical error and should be able to be performed by most microbiology laboratories. Presenter: Dr. Richard Bunter, Microbiology Department, SVH.
Presenter; Dr James Hurley, Microbiology Department, SV H
ABSTRACTS
RAPID DETECTION AND MOLECULAR CHARACTERIZATION OF AUSTRALIAN HUMAN RICKE'ITSIAL ISOLATES. Baird R W*, Ross B, Stenos J, Dwyer B. Clinical Pathology Laboratories, Fairfield Hospital, Fairfield, Victoria, 3078. Australia has a number of unique rickettsial diseases including Queensland Tick Typhus. More recently, Flinders Island Spotted Fever ( FISF ) of presumed rickettsial origin has been described. We have studied Australian human rickettsial disease from 2 perspectives. Firstly, we have detected rickettsial DNA in blood samples of 2 patients with FISF by DNA amplificationutilizing the Polymerase Chain Reaction. Confirmation of the rickettsial origin of the amplified DNA was made by hybridizationwith a rickettsiaspecific DNA probe. This probe, made from the genus-common 17 kilodalton antigen of Rickettsia australis has been fully characterised and incorporates non-radioactive detection of target molecules. We aim to develop this method to provide rapid diagnosis of rickettsia1 disease Secondly, by applying the molecular typing technique of ribosomal DNA hybridization we have investigated the DNA relatedness of Australian human rickettsial isolates from Queensland Tick Typhus and presumptive human rickettsial isolates from RSF. Digestion of purified rickettsial chromosomal DNA from human isolates with the restriction endonuclease Eco R1 revealed specific differences between rickettsial isolates from patients with Queensland Tick Typhus and FISF when probed with ribosomal DNA. Ongoing studies are in progress to establish the relatedness of these isolates. Presenter: Dr. Robert Baird, Fairfield Hospital
CULTURE POSITIVE TUBERCULOSIS: A 28 YEAR REVIEW. J.C. Hurley, E.M. Uren & J.H. Andrew, Microbiology Department, SLVincent's Hospital, Melbourne. Between 1962 and 1989, 36,000 specimens were submitted to the microbiology laboratory for examination for Mycobactefium tuberculosis (MTB). The annual number of specimens has increased from around 1,000 to 1,900 per annum over this period. We reviewed the types of specimens submitted and found a greater yield from gastric aspirate and tissue specimens in comparison to other specimens. Demographic information on the patients that were found to be culture positive for MTB was obtained from SVH medical records and notification records (through the Tuberculosis program, Health Department, Victoria). An increase in the numbers of patients whose country of birth was in Asia or the Mediterranean area was noted and these patients accounted for the majority of tuberculosis in the 1980's. The patients from these countries present at a younger age, with a higher proportion of extra-pulmonary disease and a higher proportion of drug resistant disease than patients born in Australia or Europe. Presenter; Dr James Hurley, Microbiology Department, SV H
PENICILLIN TOLERANCE IN VWDANS STREPTOCOCCI CAUSING ENDOCARDITIS - A NOVEL DETECTION METHOD EMPLOYING THE "E-TEST"
R. Bunter, K. Stockman, J. Downes, J. H. Andrew, Department of Microbiology, St. Vincent's Hospital, Melbourne, Vic. 3065. Viridans streptococci (VS) causing bacterial endocarditis are frequently tolerant to penicillin (MIC:MBC ratio 232) and a treatment combination of a penicillin and an aminoglycoside is commonly used. If the absence of tolerance could be simply and rapidly determined, there is an option of ceasing or shortening the duration of aminoglycoside therapy in patients where toxicity from this group of drugs is of concern. Determination of the MIC and MBC of VS causing endocarditis has traditionally required labour intensive broth dilution methods. MBC estimations have been poorly standardised are subject to technical error and available in few laboratories. A new method, the "E-Test" (AB Biodisk) allows the simple and accurate determination of MIC's. A modification of this method to enable the determination of tolerance was developed. Following estimation of the MIC residual penicillin was inactivated with Beta-lactamase delivered by a measured dose spray, the plate reincubated and surviving organisms in a specified area counted. Forty VS isolates from cases of proven endocarditis were examined for tolerance using the "E-Test" and traditional micro and macro broth dilution methods (NCCSLS M7-T2 and M26-P). The "E-Test" method compared very favourably, was relatively non-labour intensive, less subject to technical error and should be able to be performed by most microbiology laboratories. Presenter: Dr. Richard Bunter, Microbiology Department, SVH.