Scaiid J Infect Dis 24: 691-696, 1992
Diagnosis of Human Parvovirus B19 Infections by Polymerase Chain Reaction
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T I N 0 F. SCHWARZ', G U N D U L A J A G E R ' , WOLFGANG HOLZCiREVE' and MICHAEL ROGGENDORF" From the ' M U Xvnn Pertenkofer Institute for Hygiene and Medicul Micrnbiologv. Ludwig Muximilions University, Munich, 'Department of Gynetwlogy and Obstetrics. Wr.~rf~ilische M'ilhelms University, Munster. and the -'Institute of Virologv. University ('tinic.. D s e n . Germtiny
The polymerase chain reaction (PCR) was used for detecting parvovirus B19 DNA in clinical specimens. A pair of oligonucleotide primers spanning the Pstl-fragment of the B19 virus genome was used for PCR, and a PCR product of 727 bp was amplified. B19 virus DNA was detected in all sera ( n = 2 6 ) of individuals in the incubation period and acute phase of infection. PCR was useful for detecting viral B19 DNA in amniotic fluid and fetal blood of hydropic fetuses, confirming fetal B19 virus infection. T . F . Schwarz, MD, Max von Pettenkofer Institute fur Hygiene and Medical Microbiology, Ludwig Maximilians University, Pettenkofer Street Ya, 0-8000 Miinchen 2 , Germany
INTRODUCTION Human parvovirus B19. first described by Cossart et al. (1) causes erythema infectiosum (2). In patients with underlying chronic haemolytic anaemia, such as sickle cell anaemia, thalassaemia and spherocytosis, B19 virus infection leads to transient aplastic crisis (3). Chronic bone marrow aplasia has been observed in patients with hereditary or acquired immunodeficiency syndromes, and in these patients viraemia was prolonged and B19 virus persisted (4.5). In pregnancy, B19 virus can be transmitted transplacentally, leading to hydrops fetalis and fetal loss ( 3 , h ) . Acute B19 virus infection i s sometimes associated with SchonleinHenoch purpura (7). Bl9 virus has been identified in clinical samples of healthy individuals during acute infection by electron microscopy, and viral antigen was detected by radio (RIA)- and enzyme-linked irnmunosorbent assays (ELISA) and counter-immunoelectrophoresis (K- 10). For detecting B19 virus using these methods, approximately lo'-' virus particles/ml must be present. Several hybridization techniques for detecting B19 virus D N A in clinical specimens have been described using B19 virus D N A probes labelled with different reagents, such as "P, digoxigenin and biotin as well as chemiluminescence (10-14). The sensitivity of B19 virus DNA hybridization was equivalent to the detection of approximately 1[y virus particleshl. In patients with chronic B19 virus infections, more sensitive methods for detecting B19 virus genomes are needed, and using the polymerase chain reaction ( P C R ) , 1-10 fg of viral DNA (equivalent to 10-100 viral B19 genomes) were detected in clinical materials in different studies (15-18). We report here the use of PCR for detecting B19 virus D N A in clinical specimens, such as acute phase sera and materials associated with hydrops fetalis. MATERIALS AND METHODS Clinical specimens For evaluating PCR for detecting B19 virus DNA, u total of 52 clinical specimens were tested. These samples were:
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692 T. E Schwarz et al.
Scand J Infect Dis 24
I . Sera of patients (n = 6 ) in the viraemic phase of B19 virus infection positive for viral DNA by dot blot hybridization, and sera of exanthematic patients ( n = 20) with acute B19 virus infection, negative for viral DNA by hybridization but positive for anti-B19 IgM, as determined by ELISA. 2. Specimens of 2 cases of hydrops fetalis. The first case was a woman who had exanthema of unknown etiology 10 weeks before onset of hydrops. Serum taken at onset of hydrops was strongly positive for anti-B19 IgG but negative for IgM and viral DNA. Additionally, a fetal serum obtained by cordocentesis was negative for anti-BIY IgG and IgM by ELISA, but was very weakly positive for B19 virus DNA by dot blot hybridization. In the second case B19 virus infection was serologically confirmed by demonstrating anti-B19 IgM 6 weeks prior to onset of hydrops. To confirm fetal B19 virus infection. we tested amniotic fluid ( n = 1) and fetal serum (n = l), which were negative for anti-B19 IgM but positive for IgG as determined by ELISA (lo), and additionally, erythrocyte concentrate ( n = 1) used for intrauterine transfusion by PCR. 3. Sera (n = 20) of individuals negative for viral DNA by dot blot hybridization and anti-B19 IgM and IgG determined by ELISA with no evidence of recent contact to B19.
Positive control was a BIY virus-positive plasma (B19-MII) positive for Bl9 virus DNA by dot blot hybridization and negative for B19 virus-specific antibodies, and as negative controls, 2 sera negative for B19 virus DNA and antibodies were used.
Primers
Based on the published B19 virus DNA sequence (19), a pair of primers located in the VP2 region was selected for amplification by PCR. The two oligonucleotides were made on a DNA synthesizer (Applied Biosystems, Inc., Foster City, CA, USA) and purified with oligonucleotide purification columns supplied by the manufacturer. The sequence of primer A was 5’-AGC ATG ACT TCA GTT AAT TC-3’. corresponding to base pairs 3122-3141 of B19 virus DNA. and of primer 8 was 5’-GAT TGT ACA TIT CAT AAA AG-3’, corresponding to the antisense to base pairs 3868-3887. The two primers spanned the Pstl-fragment of the 819 virus genome and a length of 727 bp was calculated. DNA exfraction
500 PI of each specimen were digested with proteinase K , DNA extracted with phenollchloroform and precipitated with ethanol, lyophilized and resuspended in 50 pl of distilled water, according to standard procedures (20).
PCR The method used is essentially similar to the method described by Saiki et al. (21). Amplification was done in 50 pl reaction volumes. To 1 kl of extracted and resupended DNA, 49 pl of reaction mixture consisting of a total of 15 pI of Taq-buffer (Boehringer Mannheim, Mannheim, Germany), 24 pI of dNTP (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM TTP in distilled water), 101 pI of H,O, 1.6 pI T‘aq polymerase (1.5 Ufpl), 3 pl (0.06 pg/pl) of primer A and 3 pI (0.06 pgipl) of primer B, were added. The reaction mixtures were overlaid with mineral oil. The DNA amplification was carried out on a programmable heating block (Coy. Frobel, Lindau, Germany). The temperature and time regime used was as follows: 10 min at 95°C. followed by 35 cycles of 2 min 95°C. 2 min 37°C and 2 min 72°C. After the last cycle, the samples were kept for 10 min at 72°C and then stored at 4°C until performance of gel electrophoresis. Previously, we found that 42 fg of B19-DNA were detectable after 35 cycles by hybridization using cloned viral B 19-DNA (pGemPTM1; 10) as substrate. Southern blot analysis
10 pl of each PCR sample were analysed on an 1.8% agarose gel by electrophoresis in TBE buffer (80 mM Tris-borate, 1 mM EDTA pH 8.0) containing 0.5 pg/ml ethidium bromide. DNA was then transferred on to nitrocellulose (Schleicher & Schiill, Dassel, Germany) and the Southern blot performed according to standard protocols (20). Hybridization of the PCR products was performed as dcscribed previously, using a nick-translated 32P-labelled700 bp Pstl-fragment of the B19 virus genome (10).
RESULTS 46 sera of patients in the incubation period (n = 6) and acute phase (n = 20) of B19 virus
BIY P CR ifiagnosrs 693 Part~oviri~s
Scmd J lntcct Dia 24
Table I. Detection of viral B19 D N A and anti-B1Y 1gM in sera of pati~ntsin the incubat~on period (n = 6 ) , acute phase ( n = 20) of B19 inft'ctinti, and individiials ( n = 20) negatrve for B19 markers
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Scra
Incub. period ( n = 6) Acute phase ( N = 2 0 ) Individuals neg. for B1Y markers ( n = 20)
Anti-B 19 IgM-positive
PCRpositive
0
0 20
6 20
0
0
0
Dot blot hybridizationpositive 6
infection and of individuals negative for B19 markers ( n = 20) were tested by PCR. B19 virus D N A was amplified in all sera of the incubation period and acute phase by PCR, whereas no B19-DNA was amplified in sera negative for BlY markers (Table I ) . The amplified PCR products had a size of 727 bp. Specificity of PCR was demonstrated by Southern blot (data not shown). Fetal B1Y virus infection was demonstrated in both cases of hydrops fetalis. As shown in Fig. I , B19 DNA was detected only in amniotic fluid (lane 5) of case 1 . whereas no B19 D N A was amplified in maternal blood (lane 3 ) and fetal blood (lane 4). We could not detect viral D N A in fetal blood (lane 4) by PCR. although this specimen was weakly positive for B19 virus D N A by dot blot hybridization. Rcsults of analysis of specimens of case 2 are shown in Fig. 2. Fetal B 19 virus infection was confirmed in the hydropic fetus by demonstrating B l 9 virus DNA in maternal (lane 8) and fetal (lane 7) blood taken after onset of hydrops as well as in amniotic fluid (lane 5) by PCR. DISCUSSION PCR techniques have become widely used for detecting various infectious agents (22,23). In this study, we show that B1Y virus DNA can be detected by PCR in sera from the incubation period and acute phase of B19 virus infection. and fetal B19 virus infection can be confirmed in cases where diagnostic interpretation of serological results is otherwise difficult. Different Fig. 1. Ethidium-bromide stained agarose gel of PCR products of clinical specimens of a hydropic fetus (case 1). B19 virus DNA was not detected in maternal (lane 3 ) and fetal (lane 4) sera but was positive in amniotic fluid (lane 5). In lane 1 and 2 . 919 virus DNA-positive controls and in lane 6 and 7 DNA-negative controls are shown.
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694
T. F. Schwarz et al.
Scand J Infect Dis 21
Fig. 2. Ethidium-bromide stained agarose gel (a) and Southern blot (b) of PCR products of specimens of a hydropic fetus (case 2 ) . B1Y virus DNA was detected in amniotic fluid (lane 5 ) . fetal (lane 7) and maternal (lane 8) serum by PCR. Erythrocyte concentrate used for intrauterine transfusion (lane 6) was negative for B19 virus DNA. Lane 1 shows a BIY virus DNA-positive and lane 2 to 4-negative controls.
pairs of primers have been reported, each allowing amplification of B19 virus DNA of sizes from 102 to 1902 bp (15-18). Mapping B19 virus strains of different global origin revealed small differences in the restriction enzyme patterns (24). These minimal variations in the nucleotide sequence of B1Y virus genomes seem to have no importance for selecting oligonucleotide primers for detecting B1Y virus D N A by PCR. I n our laboratory, we use routinely the 700 bp PstI-fragment for dot blot hybridization (10) and in situ hybridization (6) with good experience. Therefore, we selected primers amplifying the Pstl-region of the B19 virus genome. T h e latency between acute maternal B19 virus infection and onset of fetal complication can be up to 12 weeks (25). In these cases, maternal anti-B19 IgM levels may have become undetectable, and serological confirmation of B1Y virus infection may be impossible. Detection of anti-B19 IgM in fetal blood confirms fetal B19 virus infection. It has been reported that in some B1Y virus-infected fetuses, anti-B19 IgM cannot be detected (26) but as shown in our 2 clinical cases, PCR is helpful in confirming fetal B1Y virus infection. In serum of the hydropic fetus (case l),B19 virus D N A could not be amplified by PCR, although it was present by dot blot hybridization. The discrepancy can be explained by the fetal blood sampling method. The needle used for cordocentesis in case 1 was moistened with heparin in contrast to the sampling method used in case 2. As it has been shown that this anticoagulant inhibits Taq polymerase by binding to D N A (27), we think that the use of heparin in fetal blood sampling could cause false-negative PCR results. Further studies are necessary to evaluate the inhibitory effect of heparin in detecting B19 D N A by PCR.
Scmd 1 lntcct DI\ 74
P m r i ~ n ~ BIG' r u ~ PCR diagrio~is 695
R o u t i n e u s e of PCR will i m p r o v e diagnosis of B19 infection a n d increase o u r u n d e r s t a n d i n g of the significance of this viral infection.
ACKNOWLEDGEMENTS
Scand J Infect Dis Downloaded from informahealthcare.com by Flinders University of South Australia on 12/28/14 For personal use only.
This study was supported by the Bundesministerium fur Forschung und Technologic. Bonn (grant no. OIKIX817). We thank B. Hottentrager and B. Heuser for excellent technic a I cIs\lstunce. ' . '
REFERENCES I . Cossart YE. Field AM, Cant B, Widdows D . Parvovirus-like particles i n human sera. Lancct I : 72-73, 1975. 2. Anaerson MJ. Lewis E, Kidd JM. Hall SM, Cohen BJ. An outbreak of erythema infcctiosum associated with human parvovirus B19 infection. J Hyg (Camb) 93: 85-03. 1984. 3. Centers for Disease Control. Risks associated with human parvovirus 810 infection. MMWR 38: 81-97. 198'9. 4. Kurtzman G. Frickhofen N. Kimball J . Jenkins DW. Nienhuis AW. Young NS. Pure red-cell aplasia of 10 years' duration due to persistcnt parvovirus B 19 infection and i t \ cure with immunoglobulin therapy. N Engl J Med 321: 519-523. 1989. 5 . Frickhofen N, Abkowitz JL, Safford M, Berry JM. Antunez-de-Mayolo J . Astrow A. Cohcn R. Halperin 1. King L, Mintzer D. Cohen B, Young NS. Persistent BIY parvovirus infection in patients infected with human immunodeficiency virus type I (H[V- I ) : A treatable cause o f anemia in AIDS. Ann Intern Med 113: 926033. 1990. 6. Schwdrz TF. Nerlich A , Hottentrager B. Jager G . Wicst 1, Kantimm S, Roggendorf H. Schultz M. Gloning KP. Schramm T. Holzgreve W, Roggendorf M. Parvovirus B19 infection of the fetus Histology and in situ hybridization. Am J Clin Pathol 96: 121-126. 1991 7. Lefere JJ, Courouce A M , Muller JP, Clark M. Soulier JP. Human parvovirus and purpura. Lancet 2: 730, 1985. 8. Cohen BJ, Mortimer PP, Pereira MS. Diagnostic assays with monoclonal antibodies for the human serum parvovirus-like virus (SPLV). J Hyg (Carnb) Y l : I l 5 1 3 0 . 1983. 0. Cohen BJ, Field AM. Dudnadottir S, Beard S, Barbara JAJ. Blood donor Xcrcening for parvovirus B19. J Virol Methods 30: 233-238, 1990. 10. Schwarz TF. Roggendorf M. Deinhardt F. Human parvovirus B19: ELlSA and immunoblot assays. J Virol Methods 20: 155-168, 1988. 11. Anderson MJ, Jones SE. Minson AC. Diagnosis o f human parvovirus infection by dot-blot hybridization using cloned viral D N A . J Med Virol IS: 163-172. 1985. 12. Mori J , Field A M , Clewley JP. Cohen BJ. Dot blot hybridization assay of 8 1 9 virus D N A in clinical specimens. J Clin Microbiol 27: 459-464, 1989. 13. Azzi A , Zakrzewska K, Gentilomie G, Musiaiii M. Zerhini M. Detccticin of B19 parvovirus infections by a dot-blot hybridization assay using a digoxigenin-labelled probe J Virol Methods 27: 125-134. 1990. 14. Musiani M. Zerbini M. Gibellini D, Gentolomi G. Venturoli S. Gallanella G . Ferri E. Girotti S. Chemiluminescence dot blot hybridization assay for detection of human parvovirus B19 DNA in human sera. J Clin Microbiol29: 2047-2050, 1991. 15. Clewley JP. Polymerase chain reaction assay of parvovirus B19 DNA in clinical specimens. J Clin Microbiol 27: 2647-2651, 1989. 16. Salimans MMM, Holsappel S, van de Rijke FM. Jiwa NM. Raap AK. Weiland HT. Rapid detection of human parvovirus B19 DNA by dot-hybridization and the polymerase chain reaction. J Virol Methods 23: 19-28, 1989. 17. Koeh WC. Adler SP. Detection of human parvovirus BIY DNA by using the polymerase chain reaction. J Clin Microbiol 28: 65-69, 1990. 18. Frickhofen N, Young NS. Polymerase chain reaction for detection o f parvovirus €319 in immunodeficient patients with anemia. Behring Inst Mitt XS: 3 6 5 4 . 1990, 19. Shade RO. Blundell MC. Cotrnore SF, Tatersall P. Astell C. Nucleotide sequence and genonic organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis. J Virol 58: 921-936, 1986. 20. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning - A laboratory manual. 2nd ed. Cold Spring Harbour: Cold Spring Harbour Laboratory Press. 9.3SY.42. 1989. 21. Saiki RK. Gclfand D H , Stoffel S, Scharf SJ. Higuchi R. Horn GT. Mullis KB. Erlich H A .
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22. 23. 24.
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25. 26. 27.
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Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 230: 1350-1354, 1988. Sumazaki R. Motz M, Wolf H, Heinig J, Jilg W. Deinhardt F. Detection of hepatitis B virus in serum using amplification of viral DNA by means of the polymerase chain reaction. J Med Virol27: 304-308. 1989. Molitor TW, Oraveerakul K. Zhang QQ, Choi CS. Ludemann LR. Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus. J Virol Methods 32: 201-21 1. 1991. Mori J. Beattie P, Melton DW, Cohen BJ, Clewley JP. Structure and mapping of DNA of human parvovirus B19. J Gen Virol 68: 2797-2806, 1987. Bond PR. Caul EO. Usher J, Cohen BJ, Clewley JP, Field AM. Intrauterine infection with human parvovirus. Lancet 1: 448, 1986. Anderson MJ, Khousam MN, Maxwell DJ, Gould SJ, Happerfield LC, Smith WJ. Human parvovirus B19 and hydrops fetalis. Lancet 1: 535, 1988. Beutler E, Gelbart T, Kuhl W. Interference of heparin with the polymerase chain reaction. BioTechniques 9: 166, 1990.
Diagnosis of Human Parvovirus B19 Infections by Polymerase Chain Reaction
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T I N 0 F. SCHWARZ', G U N D U L A J A G E R ' , WOLFGANG HOLZCiREVE' and MICHAEL ROGGENDORF" From the ' M U Xvnn Pertenkofer Institute for Hygiene and Medicul Micrnbiologv. Ludwig Muximilions University, Munich, 'Department of Gynetwlogy and Obstetrics. Wr.~rf~ilische M'ilhelms University, Munster. and the -'Institute of Virologv. University ('tinic.. D s e n . Germtiny
The polymerase chain reaction (PCR) was used for detecting parvovirus B19 DNA in clinical specimens. A pair of oligonucleotide primers spanning the Pstl-fragment of the B19 virus genome was used for PCR, and a PCR product of 727 bp was amplified. B19 virus DNA was detected in all sera ( n = 2 6 ) of individuals in the incubation period and acute phase of infection. PCR was useful for detecting viral B19 DNA in amniotic fluid and fetal blood of hydropic fetuses, confirming fetal B19 virus infection. T . F . Schwarz, MD, Max von Pettenkofer Institute fur Hygiene and Medical Microbiology, Ludwig Maximilians University, Pettenkofer Street Ya, 0-8000 Miinchen 2 , Germany
INTRODUCTION Human parvovirus B19. first described by Cossart et al. (1) causes erythema infectiosum (2). In patients with underlying chronic haemolytic anaemia, such as sickle cell anaemia, thalassaemia and spherocytosis, B19 virus infection leads to transient aplastic crisis (3). Chronic bone marrow aplasia has been observed in patients with hereditary or acquired immunodeficiency syndromes, and in these patients viraemia was prolonged and B19 virus persisted (4.5). In pregnancy, B19 virus can be transmitted transplacentally, leading to hydrops fetalis and fetal loss ( 3 , h ) . Acute B19 virus infection i s sometimes associated with SchonleinHenoch purpura (7). Bl9 virus has been identified in clinical samples of healthy individuals during acute infection by electron microscopy, and viral antigen was detected by radio (RIA)- and enzyme-linked irnmunosorbent assays (ELISA) and counter-immunoelectrophoresis (K- 10). For detecting B19 virus using these methods, approximately lo'-' virus particles/ml must be present. Several hybridization techniques for detecting B19 virus D N A in clinical specimens have been described using B19 virus D N A probes labelled with different reagents, such as "P, digoxigenin and biotin as well as chemiluminescence (10-14). The sensitivity of B19 virus DNA hybridization was equivalent to the detection of approximately 1[y virus particleshl. In patients with chronic B19 virus infections, more sensitive methods for detecting B19 virus genomes are needed, and using the polymerase chain reaction ( P C R ) , 1-10 fg of viral DNA (equivalent to 10-100 viral B19 genomes) were detected in clinical materials in different studies (15-18). We report here the use of PCR for detecting B19 virus D N A in clinical specimens, such as acute phase sera and materials associated with hydrops fetalis. MATERIALS AND METHODS Clinical specimens For evaluating PCR for detecting B19 virus DNA, u total of 52 clinical specimens were tested. These samples were:
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692 T. E Schwarz et al.
Scand J Infect Dis 24
I . Sera of patients (n = 6 ) in the viraemic phase of B19 virus infection positive for viral DNA by dot blot hybridization, and sera of exanthematic patients ( n = 20) with acute B19 virus infection, negative for viral DNA by hybridization but positive for anti-B19 IgM, as determined by ELISA. 2. Specimens of 2 cases of hydrops fetalis. The first case was a woman who had exanthema of unknown etiology 10 weeks before onset of hydrops. Serum taken at onset of hydrops was strongly positive for anti-B19 IgG but negative for IgM and viral DNA. Additionally, a fetal serum obtained by cordocentesis was negative for anti-BIY IgG and IgM by ELISA, but was very weakly positive for B19 virus DNA by dot blot hybridization. In the second case B19 virus infection was serologically confirmed by demonstrating anti-B19 IgM 6 weeks prior to onset of hydrops. To confirm fetal B19 virus infection. we tested amniotic fluid ( n = 1) and fetal serum (n = l), which were negative for anti-B19 IgM but positive for IgG as determined by ELISA (lo), and additionally, erythrocyte concentrate ( n = 1) used for intrauterine transfusion by PCR. 3. Sera (n = 20) of individuals negative for viral DNA by dot blot hybridization and anti-B19 IgM and IgG determined by ELISA with no evidence of recent contact to B19.
Positive control was a BIY virus-positive plasma (B19-MII) positive for Bl9 virus DNA by dot blot hybridization and negative for B19 virus-specific antibodies, and as negative controls, 2 sera negative for B19 virus DNA and antibodies were used.
Primers
Based on the published B19 virus DNA sequence (19), a pair of primers located in the VP2 region was selected for amplification by PCR. The two oligonucleotides were made on a DNA synthesizer (Applied Biosystems, Inc., Foster City, CA, USA) and purified with oligonucleotide purification columns supplied by the manufacturer. The sequence of primer A was 5’-AGC ATG ACT TCA GTT AAT TC-3’. corresponding to base pairs 3122-3141 of B19 virus DNA. and of primer 8 was 5’-GAT TGT ACA TIT CAT AAA AG-3’, corresponding to the antisense to base pairs 3868-3887. The two primers spanned the Pstl-fragment of the 819 virus genome and a length of 727 bp was calculated. DNA exfraction
500 PI of each specimen were digested with proteinase K , DNA extracted with phenollchloroform and precipitated with ethanol, lyophilized and resuspended in 50 pl of distilled water, according to standard procedures (20).
PCR The method used is essentially similar to the method described by Saiki et al. (21). Amplification was done in 50 pl reaction volumes. To 1 kl of extracted and resupended DNA, 49 pl of reaction mixture consisting of a total of 15 pI of Taq-buffer (Boehringer Mannheim, Mannheim, Germany), 24 pI of dNTP (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM TTP in distilled water), 101 pI of H,O, 1.6 pI T‘aq polymerase (1.5 Ufpl), 3 pl (0.06 pg/pl) of primer A and 3 pI (0.06 pgipl) of primer B, were added. The reaction mixtures were overlaid with mineral oil. The DNA amplification was carried out on a programmable heating block (Coy. Frobel, Lindau, Germany). The temperature and time regime used was as follows: 10 min at 95°C. followed by 35 cycles of 2 min 95°C. 2 min 37°C and 2 min 72°C. After the last cycle, the samples were kept for 10 min at 72°C and then stored at 4°C until performance of gel electrophoresis. Previously, we found that 42 fg of B19-DNA were detectable after 35 cycles by hybridization using cloned viral B 19-DNA (pGemPTM1; 10) as substrate. Southern blot analysis
10 pl of each PCR sample were analysed on an 1.8% agarose gel by electrophoresis in TBE buffer (80 mM Tris-borate, 1 mM EDTA pH 8.0) containing 0.5 pg/ml ethidium bromide. DNA was then transferred on to nitrocellulose (Schleicher & Schiill, Dassel, Germany) and the Southern blot performed according to standard protocols (20). Hybridization of the PCR products was performed as dcscribed previously, using a nick-translated 32P-labelled700 bp Pstl-fragment of the B19 virus genome (10).
RESULTS 46 sera of patients in the incubation period (n = 6) and acute phase (n = 20) of B19 virus
BIY P CR ifiagnosrs 693 Part~oviri~s
Scmd J lntcct Dia 24
Table I. Detection of viral B19 D N A and anti-B1Y 1gM in sera of pati~ntsin the incubat~on period (n = 6 ) , acute phase ( n = 20) of B19 inft'ctinti, and individiials ( n = 20) negatrve for B19 markers
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Scra
Incub. period ( n = 6) Acute phase ( N = 2 0 ) Individuals neg. for B1Y markers ( n = 20)
Anti-B 19 IgM-positive
PCRpositive
0
0 20
6 20
0
0
0
Dot blot hybridizationpositive 6
infection and of individuals negative for B19 markers ( n = 20) were tested by PCR. B19 virus D N A was amplified in all sera of the incubation period and acute phase by PCR, whereas no B19-DNA was amplified in sera negative for BlY markers (Table I ) . The amplified PCR products had a size of 727 bp. Specificity of PCR was demonstrated by Southern blot (data not shown). Fetal B1Y virus infection was demonstrated in both cases of hydrops fetalis. As shown in Fig. I , B19 DNA was detected only in amniotic fluid (lane 5) of case 1 . whereas no B19 D N A was amplified in maternal blood (lane 3 ) and fetal blood (lane 4). We could not detect viral D N A in fetal blood (lane 4) by PCR. although this specimen was weakly positive for B19 virus D N A by dot blot hybridization. Rcsults of analysis of specimens of case 2 are shown in Fig. 2. Fetal B 19 virus infection was confirmed in the hydropic fetus by demonstrating B l 9 virus DNA in maternal (lane 8) and fetal (lane 7) blood taken after onset of hydrops as well as in amniotic fluid (lane 5) by PCR. DISCUSSION PCR techniques have become widely used for detecting various infectious agents (22,23). In this study, we show that B1Y virus DNA can be detected by PCR in sera from the incubation period and acute phase of B19 virus infection. and fetal B19 virus infection can be confirmed in cases where diagnostic interpretation of serological results is otherwise difficult. Different Fig. 1. Ethidium-bromide stained agarose gel of PCR products of clinical specimens of a hydropic fetus (case 1). B19 virus DNA was not detected in maternal (lane 3 ) and fetal (lane 4) sera but was positive in amniotic fluid (lane 5). In lane 1 and 2 . 919 virus DNA-positive controls and in lane 6 and 7 DNA-negative controls are shown.
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694
T. F. Schwarz et al.
Scand J Infect Dis 21
Fig. 2. Ethidium-bromide stained agarose gel (a) and Southern blot (b) of PCR products of specimens of a hydropic fetus (case 2 ) . B1Y virus DNA was detected in amniotic fluid (lane 5 ) . fetal (lane 7) and maternal (lane 8) serum by PCR. Erythrocyte concentrate used for intrauterine transfusion (lane 6) was negative for B19 virus DNA. Lane 1 shows a BIY virus DNA-positive and lane 2 to 4-negative controls.
pairs of primers have been reported, each allowing amplification of B19 virus DNA of sizes from 102 to 1902 bp (15-18). Mapping B19 virus strains of different global origin revealed small differences in the restriction enzyme patterns (24). These minimal variations in the nucleotide sequence of B1Y virus genomes seem to have no importance for selecting oligonucleotide primers for detecting B1Y virus D N A by PCR. I n our laboratory, we use routinely the 700 bp PstI-fragment for dot blot hybridization (10) and in situ hybridization (6) with good experience. Therefore, we selected primers amplifying the Pstl-region of the B19 virus genome. T h e latency between acute maternal B19 virus infection and onset of fetal complication can be up to 12 weeks (25). In these cases, maternal anti-B19 IgM levels may have become undetectable, and serological confirmation of B1Y virus infection may be impossible. Detection of anti-B19 IgM in fetal blood confirms fetal B19 virus infection. It has been reported that in some B1Y virus-infected fetuses, anti-B19 IgM cannot be detected (26) but as shown in our 2 clinical cases, PCR is helpful in confirming fetal B1Y virus infection. In serum of the hydropic fetus (case l),B19 virus D N A could not be amplified by PCR, although it was present by dot blot hybridization. The discrepancy can be explained by the fetal blood sampling method. The needle used for cordocentesis in case 1 was moistened with heparin in contrast to the sampling method used in case 2. As it has been shown that this anticoagulant inhibits Taq polymerase by binding to D N A (27), we think that the use of heparin in fetal blood sampling could cause false-negative PCR results. Further studies are necessary to evaluate the inhibitory effect of heparin in detecting B19 D N A by PCR.
Scmd 1 lntcct DI\ 74
P m r i ~ n ~ BIG' r u ~ PCR diagrio~is 695
R o u t i n e u s e of PCR will i m p r o v e diagnosis of B19 infection a n d increase o u r u n d e r s t a n d i n g of the significance of this viral infection.
ACKNOWLEDGEMENTS
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This study was supported by the Bundesministerium fur Forschung und Technologic. Bonn (grant no. OIKIX817). We thank B. Hottentrager and B. Heuser for excellent technic a I cIs\lstunce. ' . '
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