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An investigation into the aetiological role of rotavirus in intussusception
636
ROYAI. COLLEGE
of: PATHOL(X;ISTS
mechanism>. Raised serum creatinine response.
(
OF AUSTRALASIA
Pathology (1981), 13. July
> 0.2 mnlol I ) did not affect avidity or haemagglutinating antibody
References I . SOOTHll.1.. J. F & %EWAKD, M. W. (1971): Ch/l. E.\y). I t i ~ t t ~ ~ i t9,~ d .193. 2. GEKSHWIN. M. E. & STEIS6FKG. A. D. (1974): Arthr. c t ~ dRhe~tt~I. 17, 947 954. 3. LFos. S . A. t't (11. (1977): .Art/rr. crnd R h w m . 20, 23-28. IMMUNODIAGNOSIS OF MUSCLE DISEASE: USE OF ANTI AChR AND OTHER AUTO ANTIBODIES
R. L. DAWKINS. M. GARLWI'. P. K 4 s . B. L. MCDONALD & P. J. ZILKO Depurrments qfClinicu/ H o . ~ ! ~ i (t t~i itd/ QI~CVII Ef'izuherlr II M&cd C6wIri~.Unii*ersit!,of Wes1ei.n '4 ustrdiu, Peril1 Mqasthenia gracis ( M G ) is an autoimmune disease characterized by neuromuscufar block due to an autoantibody to the rtcet>lcholine receptor (anti AChR). Experience with more than one thousand cases of M G and a similar number of controls has s h o a n that the 2 bungarotoxin assay for anti AChR provides a test of high diagnostic qecificity and sensitivit). but some aspects of the technique and interpretation are of critical importance. Results can also be useful in monitoring activity and in assessing prognosis. Pure ocular MG is not associated with anti AChR. Thynioma associated with M C should beconsidered if there is also an autoantibody to striated muscle (anti Str), but again details of the technique and interpretation are important. HLA typing may have some limited value. Polym>ositis IS a heterogeneous condition which can be subclassified by testing for autoantibodies to nuclear aiitigens. The antibod) to ribonuclear protein (anti RNP) is especially valuable in this regard. Anti R N P is often associated with characteristic muscle biopsy findings. HLA typing is of little value. even though there is a significant association betaeen HLA. A I . Bh. DR3 and juvenile dermatomyositib. ~ t l l ~ ~ J l l t l OR i fol I~d~ ~Perth ,
MOLECULAR EPIDEMIOLOGY OF HERPES VtRUSES
L. M. DESILYA & J . M. WHALLEYDepurmenf qf'I,.Irologj.,Rojwl .AIesundru Hospitalfor Children, C u i n p x i o ~ i ~ ,V.S. i i . W ,rirrd Scliool of' Biologicul Sc'iencts. Mucquurie b'niiwsitj., North Rvdf. N.S. W. Herpes simplex viruses are responsible for a variety of human infections including primary gingivostomatitis, encephalitis. herpes genitalis and recurrent cold sores. While current virological techniques can generally identify isolates as herpes simplex viruses, of either type I or type 2, it has not been possible to detect the subtle intratypic differences known toexist among different virus strains. A new approach to theepidemiologyoftheseviruses comes from the use of restriction endonucleases ahich cut DNA at specific sites. In this stud>. the DNA genomes ol'a number of herpes virus isolates have been analysed by several restriction enzymes. DNA was extracted from "P labelled small-scale infected cultures, digested with one of a number of etvymes ( e . g EcoR1. Bgl 11). and the resulting fragments separated by electrophoresis in agarose gels; autoradiograph! of dried gels revealed distinctive patterns of bands corresponding to DNA fragments of specific size. Highly reproducible results to date show ( I ) a clear and characteristic difference between the DNAs of HSV-1 and HSV-2 isolates enabling an unambiguous identification of virus type to be made, and (2) certain variations in band patterns among generally similar HSV-I isolates. These intratypic variations can be used to associate virus strains with particular sources or epidemics. and possibly also t o particular clinical manifestations. AN INVESTIGATION INTO THE AETIOLOGICAL ROLE OF ROTAVIRUS IN INTUSSUSCEPTION
D. L. ML.LCAHY. K . R. KAhlATH, L. M. DE SILVA, M. J. CLCONAN & S. HODGES Departnienrs (d G'o.rtroetitrro/, mid 1 ;iro/o~qj..Ro!d Ale.wridro HospituI for Children. Cunzpcrdown, N.S. W.: Depurtinmt of' P'irologj.. Prince Henr! Hospital,Lirrle &I., N.S. W . und Department ofkficrohiology, Rql (11 Prince .4lfid Ho.spi~ul.Cotitperdown. N . S . W.
Viruses have been incriminated as aetiological agents in intussusception. The role of rotavirus was therefore studied in a two-part inLestigation: I . Retrospecriw epiderniologicu/ stuclj.. The records o f children admitted to the Royal Alexandra Hospital for Children during the period 1975-~1978with acute intussusception and acute gastroenteritis were studied. N o cvidence of a positive correlation between the incidence of the t w o conditions was obtained.
ABSTRACTS OF ANNUAL MEETING
1980 637
2. A prospective study of rotavirus infection in children with acute intussusception. A total of 24 children admitted during the period February to July, 1979 (inclusive) with acute intussusception were investigated for rotavirus excretion by electron microscopy, ELISA and immunofluorescence. In addition, serological evidence of a response to rotavirus was sought in acute and convalescent sera. Only 2 of the 24 cases demonstrated evidence of concurrent rotavirus infection. No firm evidence of an aetiological role for rotavirus in intussusception was thus obtained in this study.
MICROBIOLOGY
COMPARISON OF AMINOGLYCOSIDE ASSAYS
V. G. SINICKAS*, D. YAU*& G. V. WILLIAMS***Biochemistry Department and **Microbiology Department, Royal Prince Alfred Hospital, Camperdown. N.S. W . Aminoglycosides are an important group of antibiotics but possess significant potential for toxicity. Serum levels are useful in making appropriate dosage alterations to avoid toxicity and obtain therapeutic concentrations. Rapid estimations are particularly helpful in patients with compromised renal function. In this respect we evaluated two methods of assaying serum gentamicin, high pressure liquid chromatography (HPLC-E) and Emit, and compared them with bioassay. HPLC-E with post-column derivatization requires sample preparation utilizing a cation exchange column, CMSephadex (G 25) to extract aminoglycoside from serum, followed by 15 ,u1 injection of the prepared sample onto a C18 uBondapak reverse phase column. The post-column derivatization utilizes ortho-phthaldialdehyde as the fluorescent label and the reaction occurs in a 2 m delay coil. Fluorescence is measured at 340 nm excitation and 418 nm emission The Emit-gentamicin assay is an ii?munoassay in which anti-gentamicin antibodies inactivate a gentamicinlabelled enzyme. Free gentamicin in the serum sample competes with the gentamicin-enzymecomplex for binding to the anti-gentamicin antibody, thereby decreasing the amount of inactivation of enzyme-labelled gentamicin. The unbound gentamicin-enzymecomplex will act upon the substrate glucose-6-phosphate with the reduction of NAD to NADH resulting in a spectrophotometric change when measured at 340 nm. The bioassay was performed in duplicate by standard disc diffusion technique using Oxoid agar No. 5 with B. cereus or E. hafnia as the test organisms. The diameter of each zone of inhibition was measured after incubation at 3 5 T for 16 h. Comparing the methods there was good correlation between the following: HPLC-E and bioassay; r = 0.92, Y = 1.06~+0.08; Emit and bioassay; r = 0.90, Y = 0 . 9 8 ~+0.59; and HPLC-E and Emit; r = 0.90, Y = 0 . 9 4 ~ +O. 13. Results were obtained within 1 h for Emit; HPLC-E required 3 h. Within-day precision (CV) for the rapid tests was 5% for Emit and 3% for HPLC-E. The Emit and HPLC-E systems did not show any interference from tobramycin, amikacin, co-trimoxazole, cephalosporins and penicillins. Further development of HPLC-E has allowed assays for tobramycin and amikacin.
THE INTERPRETATION OF MICROBIOLOGY REPORTS BY CLINICIANS
V. P. ACKERMAN*, R. C. PRITCHARD*, E. REISS-LEVY**, R. MuNRo***, P. 3. MCDONALD? & A. L E *Royal ~ North Shore Hospital, St. Leonards, N.S. W.: **St. George Hospital, Kogarah, N.S. W.;** Westmead Hospital, Wentworthville, N . S . W.; tF1inder.s Medical Centre, Bedford Park, S.A. and ??University of New South Wales, Sydney Microbiologists know very little about the effectiveness of their reports as a means of communication. We have attempted to investigate this subject by circulating typical reports to the medical staffs of 4 teaching hospitals. The reports were designed to enable us to assess the comprehension of common microbiological terms and to answer certain questions such as the effect of comments and of including antibiotic sensitivities where indications for antibiotic therapy were not definite. Several of the reports were thus prepared in 2 versions, e.g. the presence of a staphylococcus in sputum or a Salmonella in faeces was recorded with or without sensitivities. The need for explanatory comments in many ordinary reports was confirmed. The effect of reporting sensitivitieswas not clearcut. In the case of the staphylococcus in sputum this made no difference, whereas significantly more clinicians recommended antibiotic therapy for the Salmonella gastroenteritis when sensitivity results appeared in the report. A number of other findings are discussed.
ROYAI. COLLEGE
of: PATHOL(X;ISTS
mechanism>. Raised serum creatinine response.
(
OF AUSTRALASIA
Pathology (1981), 13. July
> 0.2 mnlol I ) did not affect avidity or haemagglutinating antibody
References I . SOOTHll.1.. J. F & %EWAKD, M. W. (1971): Ch/l. E.\y). I t i ~ t t ~ ~ i t9,~ d .193. 2. GEKSHWIN. M. E. & STEIS6FKG. A. D. (1974): Arthr. c t ~ dRhe~tt~I. 17, 947 954. 3. LFos. S . A. t't (11. (1977): .Art/rr. crnd R h w m . 20, 23-28. IMMUNODIAGNOSIS OF MUSCLE DISEASE: USE OF ANTI AChR AND OTHER AUTO ANTIBODIES
R. L. DAWKINS. M. GARLWI'. P. K 4 s . B. L. MCDONALD & P. J. ZILKO Depurrments qfClinicu/ H o . ~ ! ~ i (t t~i itd/ QI~CVII Ef'izuherlr II M&cd C6wIri~.Unii*ersit!,of Wes1ei.n '4 ustrdiu, Peril1 Mqasthenia gracis ( M G ) is an autoimmune disease characterized by neuromuscufar block due to an autoantibody to the rtcet>lcholine receptor (anti AChR). Experience with more than one thousand cases of M G and a similar number of controls has s h o a n that the 2 bungarotoxin assay for anti AChR provides a test of high diagnostic qecificity and sensitivit). but some aspects of the technique and interpretation are of critical importance. Results can also be useful in monitoring activity and in assessing prognosis. Pure ocular MG is not associated with anti AChR. Thynioma associated with M C should beconsidered if there is also an autoantibody to striated muscle (anti Str), but again details of the technique and interpretation are important. HLA typing may have some limited value. Polym>ositis IS a heterogeneous condition which can be subclassified by testing for autoantibodies to nuclear aiitigens. The antibod) to ribonuclear protein (anti RNP) is especially valuable in this regard. Anti R N P is often associated with characteristic muscle biopsy findings. HLA typing is of little value. even though there is a significant association betaeen HLA. A I . Bh. DR3 and juvenile dermatomyositib. ~ t l l ~ ~ J l l t l OR i fol I~d~ ~Perth ,
MOLECULAR EPIDEMIOLOGY OF HERPES VtRUSES
L. M. DESILYA & J . M. WHALLEYDepurmenf qf'I,.Irologj.,Rojwl .AIesundru Hospitalfor Children, C u i n p x i o ~ i ~ ,V.S. i i . W ,rirrd Scliool of' Biologicul Sc'iencts. Mucquurie b'niiwsitj., North Rvdf. N.S. W. Herpes simplex viruses are responsible for a variety of human infections including primary gingivostomatitis, encephalitis. herpes genitalis and recurrent cold sores. While current virological techniques can generally identify isolates as herpes simplex viruses, of either type I or type 2, it has not been possible to detect the subtle intratypic differences known toexist among different virus strains. A new approach to theepidemiologyoftheseviruses comes from the use of restriction endonucleases ahich cut DNA at specific sites. In this stud>. the DNA genomes ol'a number of herpes virus isolates have been analysed by several restriction enzymes. DNA was extracted from "P labelled small-scale infected cultures, digested with one of a number of etvymes ( e . g EcoR1. Bgl 11). and the resulting fragments separated by electrophoresis in agarose gels; autoradiograph! of dried gels revealed distinctive patterns of bands corresponding to DNA fragments of specific size. Highly reproducible results to date show ( I ) a clear and characteristic difference between the DNAs of HSV-1 and HSV-2 isolates enabling an unambiguous identification of virus type to be made, and (2) certain variations in band patterns among generally similar HSV-I isolates. These intratypic variations can be used to associate virus strains with particular sources or epidemics. and possibly also t o particular clinical manifestations. AN INVESTIGATION INTO THE AETIOLOGICAL ROLE OF ROTAVIRUS IN INTUSSUSCEPTION
D. L. ML.LCAHY. K . R. KAhlATH, L. M. DE SILVA, M. J. CLCONAN & S. HODGES Departnienrs (d G'o.rtroetitrro/, mid 1 ;iro/o~qj..Ro!d Ale.wridro HospituI for Children. Cunzpcrdown, N.S. W.: Depurtinmt of' P'irologj.. Prince Henr! Hospital,Lirrle &I., N.S. W . und Department ofkficrohiology, Rql (11 Prince .4lfid Ho.spi~ul.Cotitperdown. N . S . W.
Viruses have been incriminated as aetiological agents in intussusception. The role of rotavirus was therefore studied in a two-part inLestigation: I . Retrospecriw epiderniologicu/ stuclj.. The records o f children admitted to the Royal Alexandra Hospital for Children during the period 1975-~1978with acute intussusception and acute gastroenteritis were studied. N o cvidence of a positive correlation between the incidence of the t w o conditions was obtained.
ABSTRACTS OF ANNUAL MEETING
1980 637
2. A prospective study of rotavirus infection in children with acute intussusception. A total of 24 children admitted during the period February to July, 1979 (inclusive) with acute intussusception were investigated for rotavirus excretion by electron microscopy, ELISA and immunofluorescence. In addition, serological evidence of a response to rotavirus was sought in acute and convalescent sera. Only 2 of the 24 cases demonstrated evidence of concurrent rotavirus infection. No firm evidence of an aetiological role for rotavirus in intussusception was thus obtained in this study.
MICROBIOLOGY
COMPARISON OF AMINOGLYCOSIDE ASSAYS
V. G. SINICKAS*, D. YAU*& G. V. WILLIAMS***Biochemistry Department and **Microbiology Department, Royal Prince Alfred Hospital, Camperdown. N.S. W . Aminoglycosides are an important group of antibiotics but possess significant potential for toxicity. Serum levels are useful in making appropriate dosage alterations to avoid toxicity and obtain therapeutic concentrations. Rapid estimations are particularly helpful in patients with compromised renal function. In this respect we evaluated two methods of assaying serum gentamicin, high pressure liquid chromatography (HPLC-E) and Emit, and compared them with bioassay. HPLC-E with post-column derivatization requires sample preparation utilizing a cation exchange column, CMSephadex (G 25) to extract aminoglycoside from serum, followed by 15 ,u1 injection of the prepared sample onto a C18 uBondapak reverse phase column. The post-column derivatization utilizes ortho-phthaldialdehyde as the fluorescent label and the reaction occurs in a 2 m delay coil. Fluorescence is measured at 340 nm excitation and 418 nm emission The Emit-gentamicin assay is an ii?munoassay in which anti-gentamicin antibodies inactivate a gentamicinlabelled enzyme. Free gentamicin in the serum sample competes with the gentamicin-enzymecomplex for binding to the anti-gentamicin antibody, thereby decreasing the amount of inactivation of enzyme-labelled gentamicin. The unbound gentamicin-enzymecomplex will act upon the substrate glucose-6-phosphate with the reduction of NAD to NADH resulting in a spectrophotometric change when measured at 340 nm. The bioassay was performed in duplicate by standard disc diffusion technique using Oxoid agar No. 5 with B. cereus or E. hafnia as the test organisms. The diameter of each zone of inhibition was measured after incubation at 3 5 T for 16 h. Comparing the methods there was good correlation between the following: HPLC-E and bioassay; r = 0.92, Y = 1.06~+0.08; Emit and bioassay; r = 0.90, Y = 0 . 9 8 ~+0.59; and HPLC-E and Emit; r = 0.90, Y = 0 . 9 4 ~ +O. 13. Results were obtained within 1 h for Emit; HPLC-E required 3 h. Within-day precision (CV) for the rapid tests was 5% for Emit and 3% for HPLC-E. The Emit and HPLC-E systems did not show any interference from tobramycin, amikacin, co-trimoxazole, cephalosporins and penicillins. Further development of HPLC-E has allowed assays for tobramycin and amikacin.
THE INTERPRETATION OF MICROBIOLOGY REPORTS BY CLINICIANS
V. P. ACKERMAN*, R. C. PRITCHARD*, E. REISS-LEVY**, R. MuNRo***, P. 3. MCDONALD? & A. L E *Royal ~ North Shore Hospital, St. Leonards, N.S. W.: **St. George Hospital, Kogarah, N.S. W.;** Westmead Hospital, Wentworthville, N . S . W.; tF1inder.s Medical Centre, Bedford Park, S.A. and ??University of New South Wales, Sydney Microbiologists know very little about the effectiveness of their reports as a means of communication. We have attempted to investigate this subject by circulating typical reports to the medical staffs of 4 teaching hospitals. The reports were designed to enable us to assess the comprehension of common microbiological terms and to answer certain questions such as the effect of comments and of including antibiotic sensitivities where indications for antibiotic therapy were not definite. Several of the reports were thus prepared in 2 versions, e.g. the presence of a staphylococcus in sputum or a Salmonella in faeces was recorded with or without sensitivities. The need for explanatory comments in many ordinary reports was confirmed. The effect of reporting sensitivitieswas not clearcut. In the case of the staphylococcus in sputum this made no difference, whereas significantly more clinicians recommended antibiotic therapy for the Salmonella gastroenteritis when sensitivity results appeared in the report. A number of other findings are discussed.