- Email: [email protected]
A series of studies on the DNA diagnosis of plasmodium vivax and plasmodium falciparum malaria by PCR
Poster Sessions / Parasitology International 47 (SuppL ) (1998) 283-389
374
P-0948
P-0946 THE APPLICATION OF PCR TO THE DIAGNOSIS OF CONGENITAL TOXOPLAS~OSIS AND THE COMPARISON W I T H IFA RESULTS Kara
H*,
Ozcan K * ,
~
I~*,
Tanrxverdi
S=
*Department of Parasitology, Faculty of Medicine, University of ~ t k ' ~ r o v a , Adana. ~UREk-'Y T o x o p l a s m o s i s is a wide spread protozoon infection caused by the intracellular parasite T. gondll end generally a mild disease w i t h few or no clinical smyptoms. But. if pregnant women was infected during gestation, congenital malformation, spontaneous abortus, premature delivery and still birth occurs in fetus. In this study, 50 samples from each amnlon fluid, cord blood and mother periferal blood collected from pregnants who applied to Mersin state hospital were investigated for spesific IgM and IgG antibodies with Indirect Fluorescent Antibody (IFA) and DNA of Toxoplasma with Polymerase Chain Reaction (PCR). In mothers" sera IgM 36.0%. lgG 82.0%: in amnion fluid: IgM 24.0%. IgG 60.0%: in cord blood IgM 14.0%, IgG 54.0% were found positive. PCR positivity with Bx gen through PCR method using P~ and P4 primers was found 14.0%, 12.0% and 6.0% in mothers" sera. cord blood and amnion fluid, respectively. For diagnosi~ of congenital toxoplasmosis: PCR sensitivity and specificity was detected 16.7%, 97.4% and 22.2%. 87.5% and 57.1%. 95.3% in amnion fluid, mothers* blood and cord blood, respectively. In conclusion, it is considered that for rapid and reliable diagnosis of congenital toxoplasmosis, PCR might be used besides conventional techniques.
P.0947
g SERIES OF STdDI~ ON ~4E ~ A DIAGNOSISOF PLA~DIUM VIVAX AND PL./LqI4OOIUHFALCIP.,~UI4 M~Z~IA BY VEX
Shaoting ~ * , f i u a n j i n flaen--,Shitong fmo*,l~ing Lin*,Zhihui ~ e n = *$henzhen Sanitary and Epidemic Disease Prevention Station, S h e n z ~ and * * O e ~ t of Parasitology , Sun Yat- Sen University of 14edical science , Guangzhou,P.R.~ina Malaria i s one of the i m o e t a n t p a r a s i t i c infections
in
the
World,The improvement and ronewlent of diagnostic technique for malaria is very important in the control of the. disease. In these studies, we developed a s e r i e s new methods on the diagnosis of p.v.and p . f . m l a r i a by PCR. II The studies on detection of p.v. infections by amplifica~Ton of CT~Prepetitive gene using PER Se designedand synthesiz~)da s e t of primersspecific to P.v, t ~ gene.By P~,~2bp ~ A fragment could be amplified only from the D#iAextraction of blood from vivax malaria patients, it can detect parasite deesities as low as 2.8 paras[tes/~ ] blood (~ parnsi~20# l).l~ of 147 tg&%)aEcrc~c(~icaliy diagnosed vivax malaria cases were positive by PCR method and ~ normal blood sample were a l l negative. 2) A preliminary study on ~tactin~ P.f. based on ~R a ~ l i f l c a t i o n o f 3' _~rldf r a ~ e n t of CSP gene Based on a ~et of oli~enueleotides desie~d and synthesized by the authors,we s e e c e a s f u l | y a w l i f i e d a 24b'~ fragment at 3' end of CSP gene from 4 eulturedP, f.strains and two patients blood samples. But it coulde't a=~lified from I~IA extrectibn of two strains of p. v.,l..d.,T, gondii and the normal blo~l samples. 3) Studies on detection and identification of P.v, and p. f. by PCR In order to establish a PCR-based=ethod to detect P.v.and p .f, in blood samples in a single omplificatio~ reaction. Three primers were designed sceordir~ to the asexual st~e mlnria SSUrRNAgene, and the different size of P ~ produ(-ts w~re yield by PCR,a 314bp product for p.v.and a 43|bp product for P.f. .The P~-tmsed method was sueeesefulIy in detecting P.v. and P . f . infection in a s i n g l e PCR r e n c t i o n , ~ i c h was s p e c i f i c and s e n s i t i v e enough to detect p a r a s i t e a i a level as low as 1.08×lO -~ for P . f . and 2 . 5 6 x l f f "? for P . v . .
LACK OF EVIDENCE OF NEOSPORA INFECTION IN HUMANS
Robert F*, Toarte-Schaefer C*, Klein F** *Laboratoire de Parasitologie, H6pital Coclun, Pans. France, and "* Labomtoim D6partemental de l'Orne, Alene~n, France
Neospora caninum is a worldwide coccidian parasite, which has been confased with Toxoplasma gondii until 1984. indeed, these two parasites are morphologically and pathologically closel) related. Only some minor differences allow the distinction between cysts or tachyzoites of the tv, o species. Neosporosis causes abortion, congenital infections and neurological symptoms in a large variety of animals, which are intermediate hosts (dog, sheep, cow, mare...). Up to date, the definite host remains still unknown, but experimental transmission could be obtained by ingestion of infected tissues containing cysts. All the similarities between the two parasites, the low specificity of Neospora for intermediate hosts, and the absence of data in humans lead us to initiate a scroepidemiological study of N. caninum in humans in France. where 7oxopeasma seroprevalence is high. We investigated 500 ~ r a of immunocompetent pregnant women followed at Cochin Hospital (Paris), and for whom serological status for 7bxoplastrm ',','as s)stematically determined as recommended in France for the prevention of congenital toxoplasmosis. Antl-Toxoplasma specific IgG gere detected b) indirect immunofluorescence (Toxo-spot IFI ®, BioM~neux) and ELISA (Platelia® Toxo lgG, Sanofi-Pastear Diagnostics), and IgM b> EL]SA (Platelia® Toxo IgM, Sanofi-Pasteur Diagnostics) and immunocapture ([SAGA®, BioM~rieuxL Then, sera were tested blindly at t~o dilutions (1/20 and 1/80) by a microplate indirect immurv)fluorescence (llF) method combining anti-Neospora and anti-Toxoplasma IgG detection in separate wells of a same plate. This technique, which reveals antibodies against both parasites allowed us to eliminate cross-reactions and to determine thereby a dilution threshold. Of the sera, 157 hed a transitive Toxoplasrna serolog).. The correlation of the two Toxoplasma-l]F techniques was excellent (X2 = 439.3, p < O.(XH), thus ~altdating the mieroplate method and allowing serological interpretation for Neospora, None of the ~ sera were Ix~siti~e for Neo~pora when diluted 1/BEt. Fifty-one sera showed a v.eak fluorescence when diluted 1/2-(I. All 5] sera were also positive for anti-Ibxoplasmo specific IgG at significant uters, suggesting that Neosporafluorescence v, as likel) due to tow level-cn.~ss reactions. in conclusion, we did not point out e',idence of Neo~pora infection (or contact) in immunocompetent humans, with our serological technique. These results should be confirmed in immunodeficient humans.
!'-0949
PCR DIAGNOSIS FOR NEOSPORA CANINUM INFECTION IN ABORTED BOVINE FETUSES AND FOR TOXOPLASMA GONDII INFECTION IN HARES AND GOATS IN ITALY
Magnino S', Vigo P G*, Bandi C " , De Giuli L*" Fabb~ M', .O._en~;hLC*' *Istituto Zooprofilattico Sperimentate della Lombardia e dell'Emilia. Sezione di Pavia, Pavia, Italy and *" Istituto di Patologia Generale Veterinaria, Facolt& di Medicine Veterinaria Univers~tb, Milan Italy In the last few years, the protozoan Neospora canmum has been recognized as a major cause of abortion in cattle all over the world Besides histological and immunohistochemical examination of hssues and cultivation of the parasite from aborted fetuses and newborn calves, polymerase chain reaction (PCR)-based methods have been recently proposed !or diagnosis, mostly in experimentally infected tissues We performed a PCR assay on tissues of aborted bovine fetuses and newborn calves (n=28) employing two ohgonucteotide primers (COC-1 slightly modificated, end COC-2) specific for two regions of the rRNA gene secluences of various coccidian parasites. Tissues collected from adult hares (n=4) and goat fetuses (n=4) naturally infected and call culturepositive for Toxoplasma gond, were examined in the same way. Following DNA amplification, the products were subjected to enzymatic digestion with the restriction enzyme BsaJI DNA amplification y=elded a fragment of the expected length (294 bp) in 12 samples from bovine. 4 from hares and 4 from goat After BsaOl digestion, three bands were detected in all PCR-positive samples mn bovine specimens the bands were very close and showed up as a thick band (expected sizes: 8592.105 bp) as expected for N camnum whereas in specimens from hares and goats the bands were clearly separated (expected sizes: 153.85.43 bp) as expected for T gond# Most of the twelve PCR-bositive bovine samples belonged to herds where seropositivity for N caninum in aborted fetuses (n=8) and abortmg cows (n=2) had been detected, with Uters respectively of ;~ 1:25 and z t 640 m the indirect immunofluorescence test Our data suggest that the PCR technique can provide a faster diagnos~s for N canmum infection than the cell culture, which ~s seldom posd=ve even in immunochemistry-confirmed cases, moreover, our restr~chon analysis allows an easy differentiation between N canmumand T. gondit infections in animal tissues
374
P-0948
P-0946 THE APPLICATION OF PCR TO THE DIAGNOSIS OF CONGENITAL TOXOPLAS~OSIS AND THE COMPARISON W I T H IFA RESULTS Kara
H*,
Ozcan K * ,
~
I~*,
Tanrxverdi
S=
*Department of Parasitology, Faculty of Medicine, University of ~ t k ' ~ r o v a , Adana. ~UREk-'Y T o x o p l a s m o s i s is a wide spread protozoon infection caused by the intracellular parasite T. gondll end generally a mild disease w i t h few or no clinical smyptoms. But. if pregnant women was infected during gestation, congenital malformation, spontaneous abortus, premature delivery and still birth occurs in fetus. In this study, 50 samples from each amnlon fluid, cord blood and mother periferal blood collected from pregnants who applied to Mersin state hospital were investigated for spesific IgM and IgG antibodies with Indirect Fluorescent Antibody (IFA) and DNA of Toxoplasma with Polymerase Chain Reaction (PCR). In mothers" sera IgM 36.0%. lgG 82.0%: in amnion fluid: IgM 24.0%. IgG 60.0%: in cord blood IgM 14.0%, IgG 54.0% were found positive. PCR positivity with Bx gen through PCR method using P~ and P4 primers was found 14.0%, 12.0% and 6.0% in mothers" sera. cord blood and amnion fluid, respectively. For diagnosi~ of congenital toxoplasmosis: PCR sensitivity and specificity was detected 16.7%, 97.4% and 22.2%. 87.5% and 57.1%. 95.3% in amnion fluid, mothers* blood and cord blood, respectively. In conclusion, it is considered that for rapid and reliable diagnosis of congenital toxoplasmosis, PCR might be used besides conventional techniques.
P.0947
g SERIES OF STdDI~ ON ~4E ~ A DIAGNOSISOF PLA~DIUM VIVAX AND PL./LqI4OOIUHFALCIP.,~UI4 M~Z~IA BY VEX
Shaoting ~ * , f i u a n j i n flaen--,Shitong fmo*,l~ing Lin*,Zhihui ~ e n = *$henzhen Sanitary and Epidemic Disease Prevention Station, S h e n z ~ and * * O e ~ t of Parasitology , Sun Yat- Sen University of 14edical science , Guangzhou,P.R.~ina Malaria i s one of the i m o e t a n t p a r a s i t i c infections
in
the
World,The improvement and ronewlent of diagnostic technique for malaria is very important in the control of the. disease. In these studies, we developed a s e r i e s new methods on the diagnosis of p.v.and p . f . m l a r i a by PCR. II The studies on detection of p.v. infections by amplifica~Ton of CT~Prepetitive gene using PER Se designedand synthesiz~)da s e t of primersspecific to P.v, t ~ gene.By P~,~2bp ~ A fragment could be amplified only from the D#iAextraction of blood from vivax malaria patients, it can detect parasite deesities as low as 2.8 paras[tes/~ ] blood (~ parnsi~20# l).l~ of 147 tg&%)aEcrc~c(~icaliy diagnosed vivax malaria cases were positive by PCR method and ~ normal blood sample were a l l negative. 2) A preliminary study on ~tactin~ P.f. based on ~R a ~ l i f l c a t i o n o f 3' _~rldf r a ~ e n t of CSP gene Based on a ~et of oli~enueleotides desie~d and synthesized by the authors,we s e e c e a s f u l | y a w l i f i e d a 24b'~ fragment at 3' end of CSP gene from 4 eulturedP, f.strains and two patients blood samples. But it coulde't a=~lified from I~IA extrectibn of two strains of p. v.,l..d.,T, gondii and the normal blo~l samples. 3) Studies on detection and identification of P.v, and p. f. by PCR In order to establish a PCR-based=ethod to detect P.v.and p .f, in blood samples in a single omplificatio~ reaction. Three primers were designed sceordir~ to the asexual st~e mlnria SSUrRNAgene, and the different size of P ~ produ(-ts w~re yield by PCR,a 314bp product for p.v.and a 43|bp product for P.f. .The P~-tmsed method was sueeesefulIy in detecting P.v. and P . f . infection in a s i n g l e PCR r e n c t i o n , ~ i c h was s p e c i f i c and s e n s i t i v e enough to detect p a r a s i t e a i a level as low as 1.08×lO -~ for P . f . and 2 . 5 6 x l f f "? for P . v . .
LACK OF EVIDENCE OF NEOSPORA INFECTION IN HUMANS
Robert F*, Toarte-Schaefer C*, Klein F** *Laboratoire de Parasitologie, H6pital Coclun, Pans. France, and "* Labomtoim D6partemental de l'Orne, Alene~n, France
Neospora caninum is a worldwide coccidian parasite, which has been confased with Toxoplasma gondii until 1984. indeed, these two parasites are morphologically and pathologically closel) related. Only some minor differences allow the distinction between cysts or tachyzoites of the tv, o species. Neosporosis causes abortion, congenital infections and neurological symptoms in a large variety of animals, which are intermediate hosts (dog, sheep, cow, mare...). Up to date, the definite host remains still unknown, but experimental transmission could be obtained by ingestion of infected tissues containing cysts. All the similarities between the two parasites, the low specificity of Neospora for intermediate hosts, and the absence of data in humans lead us to initiate a scroepidemiological study of N. caninum in humans in France. where 7oxopeasma seroprevalence is high. We investigated 500 ~ r a of immunocompetent pregnant women followed at Cochin Hospital (Paris), and for whom serological status for 7bxoplastrm ',','as s)stematically determined as recommended in France for the prevention of congenital toxoplasmosis. Antl-Toxoplasma specific IgG gere detected b) indirect immunofluorescence (Toxo-spot IFI ®, BioM~neux) and ELISA (Platelia® Toxo lgG, Sanofi-Pastear Diagnostics), and IgM b> EL]SA (Platelia® Toxo IgM, Sanofi-Pasteur Diagnostics) and immunocapture ([SAGA®, BioM~rieuxL Then, sera were tested blindly at t~o dilutions (1/20 and 1/80) by a microplate indirect immurv)fluorescence (llF) method combining anti-Neospora and anti-Toxoplasma IgG detection in separate wells of a same plate. This technique, which reveals antibodies against both parasites allowed us to eliminate cross-reactions and to determine thereby a dilution threshold. Of the sera, 157 hed a transitive Toxoplasrna serolog).. The correlation of the two Toxoplasma-l]F techniques was excellent (X2 = 439.3, p < O.(XH), thus ~altdating the mieroplate method and allowing serological interpretation for Neospora, None of the ~ sera were Ix~siti~e for Neo~pora when diluted 1/BEt. Fifty-one sera showed a v.eak fluorescence when diluted 1/2-(I. All 5] sera were also positive for anti-Ibxoplasmo specific IgG at significant uters, suggesting that Neosporafluorescence v, as likel) due to tow level-cn.~ss reactions. in conclusion, we did not point out e',idence of Neo~pora infection (or contact) in immunocompetent humans, with our serological technique. These results should be confirmed in immunodeficient humans.
!'-0949
PCR DIAGNOSIS FOR NEOSPORA CANINUM INFECTION IN ABORTED BOVINE FETUSES AND FOR TOXOPLASMA GONDII INFECTION IN HARES AND GOATS IN ITALY
Magnino S', Vigo P G*, Bandi C " , De Giuli L*" Fabb~ M', .O._en~;hLC*' *Istituto Zooprofilattico Sperimentate della Lombardia e dell'Emilia. Sezione di Pavia, Pavia, Italy and *" Istituto di Patologia Generale Veterinaria, Facolt& di Medicine Veterinaria Univers~tb, Milan Italy In the last few years, the protozoan Neospora canmum has been recognized as a major cause of abortion in cattle all over the world Besides histological and immunohistochemical examination of hssues and cultivation of the parasite from aborted fetuses and newborn calves, polymerase chain reaction (PCR)-based methods have been recently proposed !or diagnosis, mostly in experimentally infected tissues We performed a PCR assay on tissues of aborted bovine fetuses and newborn calves (n=28) employing two ohgonucteotide primers (COC-1 slightly modificated, end COC-2) specific for two regions of the rRNA gene secluences of various coccidian parasites. Tissues collected from adult hares (n=4) and goat fetuses (n=4) naturally infected and call culturepositive for Toxoplasma gond, were examined in the same way. Following DNA amplification, the products were subjected to enzymatic digestion with the restriction enzyme BsaJI DNA amplification y=elded a fragment of the expected length (294 bp) in 12 samples from bovine. 4 from hares and 4 from goat After BsaOl digestion, three bands were detected in all PCR-positive samples mn bovine specimens the bands were very close and showed up as a thick band (expected sizes: 8592.105 bp) as expected for N camnum whereas in specimens from hares and goats the bands were clearly separated (expected sizes: 153.85.43 bp) as expected for T gond# Most of the twelve PCR-bositive bovine samples belonged to herds where seropositivity for N caninum in aborted fetuses (n=8) and abortmg cows (n=2) had been detected, with Uters respectively of ;~ 1:25 and z t 640 m the indirect immunofluorescence test Our data suggest that the PCR technique can provide a faster diagnos~s for N canmum infection than the cell culture, which ~s seldom posd=ve even in immunochemistry-confirmed cases, moreover, our restr~chon analysis allows an easy differentiation between N canmumand T. gondit infections in animal tissues