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Application of DNA technology for viral diagnosis
ABSTRACTS
AI'PLICAJION DIAGNOSIS
OF
DNA
TECfINOI.OC;Y
Y.E. Cossart, Deprtment of Infectirius Sydiicy NSW 2oQ6
FOR
VIRAL
Discases, University of
No area of medicine appears to be exempt from the curiosity ol
molecular biologists and the new technology has begun to change our understanding of the pathogenesis of disease fundamentally that DNA probes may even come to rival the microscope a s a symbol of m,edical research. What has molecular biology to contribute to diagnostic medicine? Some of the first applications have involved DNA probes for detection of viral genetic material in tissue and body fluids. Infection with agents which cannot be cultivated can be diagnosed with confidence by this means a n d the involvement of viruses in chronic diseases,
including tumours, can be investigated. Hepatitis B will be used as an example to demonstrate the value of molecular biology a) in diagnosis on serum samples and liver biopsies, b) to yield reagents and c) even for vaccine Discussion of the use of molecular techniques in demonstrating the role of human papillomavirus in causing cancer of the cerix will illushate potential problems in diagnostic application of recDNA methods. Finally the value of DNA polymorphism in epidemiological tracing will be addressed.
EPlOEHlOLffiY OF GONOCOCCAL DISEASE
IN HONG KONG
S.I. Egglestone. Department of Health Sclences llong Kong Polytechnic, Kowloon, Ilorig Kong.
Records as early as l8hg show that gonococcal dlsease was a cornon problem I n llong Kong particularly amongst the armed forces. llowever I n the subsequent years t h l s has varled as a result of legal control of prostitution, towards western medlclne wars changlng a t t l tudes establishment of Venereal Dlsease c l l n l c s (1628) a d antlblotlc treatment. I n the thlrteen years followlng the f l r s t use of penlclllln and Introductlon of wider social hyglene a c t l v l t i e s there was a contlnuous yearly decreasr? I n cases of gonococcal infectlon. I n 1953. 11625 cases were reported (775 per 100000 population 15 years and over) compared ulth 5096 cases I n 1965 (240 per 100000). B 1978 the lncldence had decreased to 65 per I00000 afLhough there had been two peaks in 1968 and 1972 w i t h rate? reported respectlvely a s 350 and 305 per 100000, From 1979 untll 1987 the Incidence of gonococcal Infection Increased gradually b u t decreased I n 1988 and preliminary flgures for 1989 su gest a possible substantlal further reductlon. I n 1986 491 cases (140 per 100000) were reported, a drop o r 18.52 from I987 and of these cases 872 Were men and 132 were women. A total of 89% or the cases 15.3> in occurred In persons aged less than 40 years peraons aged 30-39 years, 67.22 In persons Hged 20-29 years and 6.52 I n teenagers. Whilst the Incidence of gonococcal infection dropped among male teenagers, more teenage g i r l s were affected particularly I n the 13-16 age group. Another change noted over the last flve years h a s been the number or cases lnrected abroad. I n 1983 It was 192 and thls has risen t o 211.42 I n 1988 ulth the ;Ix most comonly Involved countrles being Hacau. China. Korea, Thailand, Phlllipines ard Taluan. The greatest Increase was from Chlna. 9 0 cases (4.9%) I n 1983 to 261 cases (25.72) I n 1988. Laboratory monltorlng of gonococcal Isolates for penlclllln reslstance has shown that the prevalence of reslstant s t r a i n s has varied over the last flve years and currently stands a t 53% for the f l r s t quarter of 1989 (602 of these are PPNG).
.
EPlDEMlOLOCY OF MULTI-RESlSTANT STAPHYLOCOCCUS AUREUS IN IlONC KONC
CHEHILUHINESCENCE AND OPSONIZATION INFECTIONS.
C.L. French*, Department of Chinese U n i v e r s i t y of Hong Kong, Kong.
J.W.H. Lawton*, K.Y. Yuen. Department of Pathology and Department of H i c r o b i o l o g y , U n i v e r s i t y of Hong Kong
Microbiology, S h a t i n , N.T.,
The Hong
[MRSA) M ~ t h i c i l l i n - r e s i s t a n t Staphylococcus aureua a r e now common i n Hong Kong, and a c c o u n t f o r 30% of a l l h o a p i t a l Staph aureua (SA) i s o l a t e s . A l l HRSA were analyaed from a new t e a c h i n g h o s p i t a l between 1984 and 1986, and 1 2 h o a p i t a l a i n Hong Kong i n l a o l a t e a of October 1985. Over 600 n o n - d u p l i c a t e HRSA were a n a l y e e d . 11 of t h e 1 2 h o a p i t a l s were a f f e c t e d by HRSA, and between 8-532 of a l l i a o l a t e a MRSA were i s o l a t e d from d e e p s i t e s of SA were HRSA. a a w e l l a a s u p e r f i c i a l , and about 30X of SA blood i s o l a t e s were MRSA. More than 20 plasmid t y p e s of HRSA were i d e n t i f i e d , b u t two kinda pre-dominated and many of t h e typee i d e n t i f i e d may be aub-typea of t h e s e two. One of t h e common typea warn n o n - t y p a b l e by phage, and t h e o t h e r r e a c t e d w i t h phages 2 9 , 77, 84 and 85. I a o l a t e a were u a u a l l y m u l t i p l y - r e a i a t a n t to several antistaphylococcal agents, especially gentamicin, erythromycin, chloranphenicol and tetracycline. HRSA i n f e c t i o n waa widespread, but Plasmid moat conmon on Burna, ITU and SCBU wards. t y p e 2 wan w i d e l y d i s t r i b u t e d i n d i f f e r e n t medical s p e c i a l t i e s , b u t plaamid t y p e 1 waa moat commonly i s o l a t e d from n e o n a t a l and p a e d i a t r i c warde. There v a a e v i d e n c e t h a t t y p e 1 waa b e i n g s p r e a d between h o a p i t a l a by p a t i e n t t r a n s f e r s w i t h i n s p e c i a l t i e s . Owing t o overcrowding, p a t i e n t t r a n s f e r s , and l a c k of I s o l a t i o n f a c i l i t i e s . t h e c o n t r o l of HRSA i n Hong Kong h o s p i t a l a w t l l be d i f f i c u l t .
IN
STREPTOCOCCAL
.
The p r o d u c t i o n of o x i d a t i v e m e t a b o l i t e s by neutrop h i l s may b e measured by chemiluminescence (CL) and t h i s is a m p l i f i e d by t h e chemical 'Luminol'. Stimul a t i o n of t h e n e u t r o p h i l o x i d a t i v e r e s p i r a t o r y b u r s t r a p i d l y f o l l o w s t h e i n t e r a c t i o n of c e l l membrane Fc and C3b r e c e p t o r s w i t h opsonized p a r t i c l e s . The Luminol-dependent CL r e s p o n s e therefore provides a s e n s i t i v e measure of t h e l e v e l of o p s o n i z a t i o n of b a c t e r i a when t h e opsonized b a c t e r i a a r e added t o n e u t r o p h i l a in s u s p e n s i o n . I n t h e present study, Strep. human n e u t r o p h i l CL r e s p o n s e s t o opsonized f a e c a l t a , and Strep. aangute were c h a r a c t e r i s e d . phorbol-myriatate-acetate (PHA) The r e s p o n s e to served a s a p o s i t i v e control. S e r a from 20 normal a d u l t s were pooled t o p r o v i d e a normal r e f e r e n c e serum f o r o p s o n i z a t i o n . I n e a c h a s s a y peak CL (mv) was measured a f t e r s t i m u l a t i o n of n e u t r o p h i l s w i t h b a c t e r i a opsonized by t h e test serum and b a c t e r i a opsonized by t h e s t a n d a r d pooled s e r u m . The 'Opsonic I n d e x ' was t h e r a t i o of t h e two peak CL values. S e r a were o b t a i n e d from p a t i e n t s w i t h s t r e p . f a e c a l i s and S t r e p . s a n g u i a i n f e c t i o n s and a l i q u o t s s t o r e d a t -7O'C p r i o r t o a s s a y . Heasurement of t h e opsonic c a p a c i t i e s of t h e s e r a towards t h e s e two S t r e p t o c o c c a l s p e c i e s and S. a u r e u a s h o u l d i n d i c a t e whether any specific opsonic defect contributed t o the development of t h e i n f e c t i o n . The a s s a y has p o t e n t i a l f o r t h e i n v e s t i g a t i o n of p a t i e n t s w i t h recurrent bacterial infection.
AI'PLICAJION DIAGNOSIS
OF
DNA
TECfINOI.OC;Y
Y.E. Cossart, Deprtment of Infectirius Sydiicy NSW 2oQ6
FOR
VIRAL
Discases, University of
No area of medicine appears to be exempt from the curiosity ol
molecular biologists and the new technology has begun to change our understanding of the pathogenesis of disease fundamentally that DNA probes may even come to rival the microscope a s a symbol of m,edical research. What has molecular biology to contribute to diagnostic medicine? Some of the first applications have involved DNA probes for detection of viral genetic material in tissue and body fluids. Infection with agents which cannot be cultivated can be diagnosed with confidence by this means a n d the involvement of viruses in chronic diseases,
including tumours, can be investigated. Hepatitis B will be used as an example to demonstrate the value of molecular biology a) in diagnosis on serum samples and liver biopsies, b) to yield reagents and c) even for vaccine Discussion of the use of molecular techniques in demonstrating the role of human papillomavirus in causing cancer of the cerix will illushate potential problems in diagnostic application of recDNA methods. Finally the value of DNA polymorphism in epidemiological tracing will be addressed.
EPlOEHlOLffiY OF GONOCOCCAL DISEASE
IN HONG KONG
S.I. Egglestone. Department of Health Sclences llong Kong Polytechnic, Kowloon, Ilorig Kong.
Records as early as l8hg show that gonococcal dlsease was a cornon problem I n llong Kong particularly amongst the armed forces. llowever I n the subsequent years t h l s has varled as a result of legal control of prostitution, towards western medlclne wars changlng a t t l tudes establishment of Venereal Dlsease c l l n l c s (1628) a d antlblotlc treatment. I n the thlrteen years followlng the f l r s t use of penlclllln and Introductlon of wider social hyglene a c t l v l t i e s there was a contlnuous yearly decreasr? I n cases of gonococcal infectlon. I n 1953. 11625 cases were reported (775 per 100000 population 15 years and over) compared ulth 5096 cases I n 1965 (240 per 100000). B 1978 the lncldence had decreased to 65 per I00000 afLhough there had been two peaks in 1968 and 1972 w i t h rate? reported respectlvely a s 350 and 305 per 100000, From 1979 untll 1987 the Incidence of gonococcal Infection Increased gradually b u t decreased I n 1988 and preliminary flgures for 1989 su gest a possible substantlal further reductlon. I n 1986 491 cases (140 per 100000) were reported, a drop o r 18.52 from I987 and of these cases 872 Were men and 132 were women. A total of 89% or the cases 15.3> in occurred In persons aged less than 40 years peraons aged 30-39 years, 67.22 In persons Hged 20-29 years and 6.52 I n teenagers. Whilst the Incidence of gonococcal infection dropped among male teenagers, more teenage g i r l s were affected particularly I n the 13-16 age group. Another change noted over the last flve years h a s been the number or cases lnrected abroad. I n 1983 It was 192 and thls has risen t o 211.42 I n 1988 ulth the ;Ix most comonly Involved countrles being Hacau. China. Korea, Thailand, Phlllipines ard Taluan. The greatest Increase was from Chlna. 9 0 cases (4.9%) I n 1983 to 261 cases (25.72) I n 1988. Laboratory monltorlng of gonococcal Isolates for penlclllln reslstance has shown that the prevalence of reslstant s t r a i n s has varied over the last flve years and currently stands a t 53% for the f l r s t quarter of 1989 (602 of these are PPNG).
.
EPlDEMlOLOCY OF MULTI-RESlSTANT STAPHYLOCOCCUS AUREUS IN IlONC KONC
CHEHILUHINESCENCE AND OPSONIZATION INFECTIONS.
C.L. French*, Department of Chinese U n i v e r s i t y of Hong Kong, Kong.
J.W.H. Lawton*, K.Y. Yuen. Department of Pathology and Department of H i c r o b i o l o g y , U n i v e r s i t y of Hong Kong
Microbiology, S h a t i n , N.T.,
The Hong
[MRSA) M ~ t h i c i l l i n - r e s i s t a n t Staphylococcus aureua a r e now common i n Hong Kong, and a c c o u n t f o r 30% of a l l h o a p i t a l Staph aureua (SA) i s o l a t e s . A l l HRSA were analyaed from a new t e a c h i n g h o s p i t a l between 1984 and 1986, and 1 2 h o a p i t a l a i n Hong Kong i n l a o l a t e a of October 1985. Over 600 n o n - d u p l i c a t e HRSA were a n a l y e e d . 11 of t h e 1 2 h o a p i t a l s were a f f e c t e d by HRSA, and between 8-532 of a l l i a o l a t e a MRSA were i s o l a t e d from d e e p s i t e s of SA were HRSA. a a w e l l a a s u p e r f i c i a l , and about 30X of SA blood i s o l a t e s were MRSA. More than 20 plasmid t y p e s of HRSA were i d e n t i f i e d , b u t two kinda pre-dominated and many of t h e typee i d e n t i f i e d may be aub-typea of t h e s e two. One of t h e common typea warn n o n - t y p a b l e by phage, and t h e o t h e r r e a c t e d w i t h phages 2 9 , 77, 84 and 85. I a o l a t e a were u a u a l l y m u l t i p l y - r e a i a t a n t to several antistaphylococcal agents, especially gentamicin, erythromycin, chloranphenicol and tetracycline. HRSA i n f e c t i o n waa widespread, but Plasmid moat conmon on Burna, ITU and SCBU wards. t y p e 2 wan w i d e l y d i s t r i b u t e d i n d i f f e r e n t medical s p e c i a l t i e s , b u t plaamid t y p e 1 waa moat commonly i s o l a t e d from n e o n a t a l and p a e d i a t r i c warde. There v a a e v i d e n c e t h a t t y p e 1 waa b e i n g s p r e a d between h o a p i t a l a by p a t i e n t t r a n s f e r s w i t h i n s p e c i a l t i e s . Owing t o overcrowding, p a t i e n t t r a n s f e r s , and l a c k of I s o l a t i o n f a c i l i t i e s . t h e c o n t r o l of HRSA i n Hong Kong h o s p i t a l a w t l l be d i f f i c u l t .
IN
STREPTOCOCCAL
.
The p r o d u c t i o n of o x i d a t i v e m e t a b o l i t e s by neutrop h i l s may b e measured by chemiluminescence (CL) and t h i s is a m p l i f i e d by t h e chemical 'Luminol'. Stimul a t i o n of t h e n e u t r o p h i l o x i d a t i v e r e s p i r a t o r y b u r s t r a p i d l y f o l l o w s t h e i n t e r a c t i o n of c e l l membrane Fc and C3b r e c e p t o r s w i t h opsonized p a r t i c l e s . The Luminol-dependent CL r e s p o n s e therefore provides a s e n s i t i v e measure of t h e l e v e l of o p s o n i z a t i o n of b a c t e r i a when t h e opsonized b a c t e r i a a r e added t o n e u t r o p h i l a in s u s p e n s i o n . I n t h e present study, Strep. human n e u t r o p h i l CL r e s p o n s e s t o opsonized f a e c a l t a , and Strep. aangute were c h a r a c t e r i s e d . phorbol-myriatate-acetate (PHA) The r e s p o n s e to served a s a p o s i t i v e control. S e r a from 20 normal a d u l t s were pooled t o p r o v i d e a normal r e f e r e n c e serum f o r o p s o n i z a t i o n . I n e a c h a s s a y peak CL (mv) was measured a f t e r s t i m u l a t i o n of n e u t r o p h i l s w i t h b a c t e r i a opsonized by t h e test serum and b a c t e r i a opsonized by t h e s t a n d a r d pooled s e r u m . The 'Opsonic I n d e x ' was t h e r a t i o of t h e two peak CL values. S e r a were o b t a i n e d from p a t i e n t s w i t h s t r e p . f a e c a l i s and S t r e p . s a n g u i a i n f e c t i o n s and a l i q u o t s s t o r e d a t -7O'C p r i o r t o a s s a y . Heasurement of t h e opsonic c a p a c i t i e s of t h e s e r a towards t h e s e two S t r e p t o c o c c a l s p e c i e s and S. a u r e u a s h o u l d i n d i c a t e whether any specific opsonic defect contributed t o the development of t h e i n f e c t i o n . The a s s a y has p o t e n t i a l f o r t h e i n v e s t i g a t i o n of p a t i e n t s w i t h recurrent bacterial infection.