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Home , Herpes simplex virus, Parvovirus B19

Relevance of , 2, and Virologic Markers in Maternal Serum for Diagnosis of Unexplained Recurrent Abortions

Maysaa El-Sayed Zaki, MD; Hossam Goda, MD

● Context.—The impact of viral during pregnan- between the RSA group and the pregnant women without cy on adverse pregnancy outcomes is not understood fully. RSA group in frequency of parvovirus IgM (84% and Objective.—To assess the frequency of parvovirus B19, 16.7%, respectively) (P Ͻ .001) and herpes simplex IgM Parvovirus B19 viremia was .(001. ؍ herpes simplex 2, and cytomegalovirus infections in rela- (40% for RSA) (P tion to late abortions, in sera from Egyptian pregnant wom- positive in 48% RSA, 2 was positive en to establish basic knowledge for future pregnancy care. in 32% RSA, and cytomegalovirus was positive in 12% In addition, to study the diagnostic value of specific im- RSA patients. For RSA patients with parvovirus viremia, the munoglobulin M (IgM) against those compared mean ؎ SD of IgM value was 78.5 ؎ 30.12 IU/mL, and ؎ with their detection by polymerase chain reaction for RSA patients with negative viremia it was 30.02 in maternal serum as a noninvasive method of laboratory 17.64 IU/mL with statistically significant difference be- diagnosis. tween both levels (P Ͻ .001). Design.—Patients were recruited at the Women’s Clinic, Conclusions.—From this study, we conclude that viral Mansoura University. One group of patients with recurrent infections with parvovirus B19 and herpes simplex 2 were spontaneous abortions (RSA) and a second group of preg- frequently associated with recurrent abortions, and careful nant women without a history of RSA were evaluated in- investigation for this condition must include evaluating cluding demographic, medical, and clinical data. Virologic these patients for the previously mentioned viruses. Sero- markers were evaluated for specific IgM and for viral DNA logic study by specific IgM for parvovirus and herpes sim- to cytomegalovirus, herpes simplex virus 2, and parvovirus plex seem to be reliable as screening tests for high-risk B19. pregnancy. Results.—There was a statistically significant difference (Arch Pathol Lab Med. 2007;131:956–960)

ome evidence suggests that intrauterine infections play patients, acute polyarthralgia syndrome in adults, hy- S a major role in the pathogenesis of early pregnancy drops fetalis, spontaneous abortion, and .4,5 loss, but the implication and prevalence of microorgan- Because B19 has been associated with a wide isms in the etiology of spontaneous abortion during the variety of clinical manifestations and some clinical fea- first trimester of pregnancy has not yet been well estab- tures of B19 infection such as or rash can be com- lished.1 mon to other , a specific laboratory identification Cytomegalovirus (CMV) and parvovirus B19 are linked of B19 is required and any diagnostic tool must consider to both late abortion and stillbirth.2,3 Parvovirus B19 in- both the type of pathology and the type of patient. In fections are associated with different clinical manifesta- immunocompetent individuals, virologic and serologic tions that vary from asymptomatic to severe symptoms. testing are complementary, whereas in immunocompro- The main clinical manifestations are erythema infectios- mised patients viral detection is the test of choice. um, transient aplastic crisis in individuals with hemoglo- Today, viral detection is generally based on direct de- binopathies, chronic anemia in the immunocompromised tection of the B19 in clinical samples.6 Another virus that could be implicated in recurrent abortion is herpes simplex. is the result of Accepted for publication October 23, 2006. infection by herpes simplex virus type 2 (HSV-2) and to a From the Departments of Clinical Pathology (Dr El-Sayed Zaki) and lesser extent HSV type 1 (HSV-1). Recently there has been Gynacology and Obstetrics (Dr Goda), Faculty of Medicine, Mansoura a rise in the prevalence of genital HSV infections in both University, Mansoura, Egypt. industrialized and developing countries. The main factors The authors have no relevant financial interest in the products or attributed to the spread of HSV include asymptomatic vi- companies described in this article. Reprints: Maysaa El-Sayed Zaki, MD, Clinical Pathology, Faculty of rus shedding and underrecognition and underdiagnosis Medicine, Mansoura University, Mansoura, 65, Egypt (e-mail: may of the disease. At the level of the individual patient, genital [email protected]). herpes is associated with significant psychological mor- 956 Arch Pathol Lab Med—Vol 131, June 2007 High-Risk Pregnancies, Viral Infections, Screening—El-Sayed Zaki & Goda bidity and complications such as neonatal herpes, the re- (221 bp) for CMV was resolved on a 1.5% agarose gel, visualized sult of of HSV from mother to baby.7 The using ethidium bromide (0.5 ␮g/mL) under ultraviolet illumi- incidence of asymptomatic cervical HSV-2 infections was nation. considerably higher in patients with a history of sponta- Multiplex Nested PCR for HSV-1/HSV-2 neous abortion with a possible etiologic connection be- tween HSV and spontaneous abortion.8 DNA was extracted with the commercially available Qiagen kit Serologic assays were not very useful for the elucidation (GmbH, Hilden). Primers were designed to bracket a well-con- served region in the DNA polymerase gene.11 Primer pair HSV- of the role of HSV in inducing spontaneous abortions, al- P1 (5Ј-GTGGTGGACTTTGCCAGCCTGTACCC-3Ј) and HSV-P2 though they indicate that the state of pregnancy predis- (5Ј-TAAACATGGAGTCCGTGTCGCCGTAGATGA-3Ј) were used 9 poses to HSV reactivation. to amplify HSV-1 and HSV-2.11 The objectives of this study were to evaluate the role of Taq (0.25 ␮L) and extracted DNA (10 ␮L) were added to each infections with parvovirus B19, HSV-2, and CMV in re- premixed supplied tube. Negative control was analyzed by add- current abortions and to study the diagnostic value of spe- ing water instead of DNA and positive control was performed cific immunoglobulin (Ig) M against those viruses com- by 5.0 ␮L of HSV-1 positive control and 5.0 ␮L of positive control HSV-2 DNA. The following program was used for the thermal pared with detecting the presence of their genomes by Њ Њ polymerase chain reaction (PCR) in maternal serum. cycle: 1 cycle at 94 C for 2 minutes, 35 cycles (94 C for 30 seconds, 56ЊC for 30 seconds, 72ЊC for 30 seconds), and 1 cycle at 72ЊC for MATERIALS AND METHODS 5 minutes. From amplified product we added 1 ␮L DNA and 0.25 ␮Lof Patients Taq polymerase in premixed tubes supplied with the kit. The Њ The patients were recruited from an obstetric outpatient clinic program used in the thermal cycles was 1 cycle at 94 C for 2 Њ Њ at the Mansoura University Hospital. Two different groups were minutes, 30 cycles (of 94 C for 30 seconds, 58 C for 30 seconds, Њ Њ evaluated. The first group (n ϭ 50) consisted of patients with 72 C for 30 seconds), and 1 cycle at 72 C for 5 minutes. Following medically unexplained recurrent spontaneous abortions (RSA) PCR, the amplicon (137 bp for HSV-1 and 100 bp for HSV-2) was (women with a history of 3 or more consecutive spontaneous resolved on a 1.5% agarose gel and visualized using ethidium ␮ abortions including abortions up to 22 gestational weeks). The bromide (0.5 g/mL) under ultraviolet illumination. second group (n ϭ 12) consisted of pregnant women without a history of RSA and with pregnancy duration of more than 32 PCR for Parvovirus B19 weeks’ gestation. The demographic, medical, and clinical data Primers were designed to bracket a well-conserved region in were collected in each case based on personal interviews and parvovirus B19. The primers were pair A (5Ј-TGT GGT AAG medical examination. AAA AAT AC-Ј3) and (5Ј-TCA TTA AAT GGA TTT-Ј3) and prim- The women signed an informed consent before they were in- er B (5Ј-GGA ACA GACTTA GAG CTT ATTC-Ј3) and (5Ј-ACC cluded in this study. The study was approved by the ethics com- CAT CCT CTC TGT TTG ACT TAG TTG CTC GTAT-Ј3). Ampli- mittee of Mansoura University. fication was done in 50-␮L reaction volume of 67mM Tris (pH

8.8), 16.6mM (NH4)2SO4, 6.7mM MgCl2, 10mM mercaptoethanol, Sera Collection 6.7␮M EDTA, 170 ␮g of bovine serum albumin per mL, 50 pmol Blood samples were obtained from each patient and centri- of each primer, and 1 U Taq polymerase overlaid with mineral fuged, and the sera were kept frozen in aliquots at Ϫ20ЊC until oil. The positive control DNA to establish the specificity of the analysis. reaction was cloned parvovirus B19 virus.12 The temperature and the time regimen used was as follows: 5 minutes at 95ЊC, 30 Serologic Study for Parvovirus B19, seconds at 50ЊC, and 35 cycles of 91ЊC for 1 minute, 50ЊC for 1 HSV-1/HSV-2, and CMV minute, and 67ЊC for 3 minutes. Following PCR, the amplicon (218 bp) was resolved on a 1.5% agarose gel and visualized using Serum samples from each patient were analyzed for qualitative ethidium bromide (0.5 ␮g/mL) under ultraviolet illumination. specific IgM for CMV, HSV-1/HSV-2 (ELISA-Equipar Via G, Fer- rari, Saronno, Italy), and parvovirus B19 IgM with quantitative Statistical Analysis positive of 13 or greater IU/mL (GmbH, Hamburg, Germany). Values were represented as means Ϯ SD, median (range), or PCR for Parvovirus B19, HSV-1/HSV-2, and CMV the number of subjects and proportions. One-way analysis of var- iance test and independent samples Student t test were used for Polymerase chain reaction was performed for each patient to group comparisons of normally distributed variables, and the detect specific DNA of CMV (Experteam Di De Bortoli Angelo Kruskal-Wallis test and Mann-Whitney U test were used for com- and CSAS, Spain), HSV-1/HSV-2, and parvovirus B19 (ABAnal- parisons of variables with skewed distribution. The chi-square ytica Svizzera PADOVA Italia, Milan, Italy). test was used to compare proportions. The receiver operator characteristic method was used to deter- PCR for CMV mine the best possible cutoff values for parvovirus IgM as a pre- DNA of CMV was extracted from a serum sample by QiAamp dictor of infection according to PCR; the curves were obtained DNA mini kit (GmbH, Hilden, Germany). For each sample, we by plotting sensitivity on the y-axis against the false-positive rate prepared the following mixture: buffer (2.5 ␮L), deoxynucleoside (1 Ϫ specificity) on the x-axis for all possible cutoff values of the triphosphates (2.5 ␮L), primer P1 (1.0 ␮L), primer P2 (1.0 ␮L), tests. From this curve, the best or optimal cutoff value was de- Taq polymerase (0.3 ␮L), distilled water (7.7 ␮L), extracted DNA termined. Significance was considered when P Ͻ .05. solution (10 ␮L), and overlaid mineral oil (30 ␮L). The PCR for detection of major immediate-early gene MIE region was per- RESULTS formed as described.10 The nested primers of mtr II, CMTR 1-5Ј- Ј Ј Fifty patients with a history of recurrent abortions and CTG TCG GTG ATG GTC TCT TC-3 and CMTR 2-5 -CCC GAC 12 pregnant women without a history of recurrent abor- ACG CGG AAA AGA AA-3Ј for the first round and CMTR 3-5Ј- TCT CTG GTC CTG ATC GTC TT-3Ј and CMTR 4-5Ј-GTG ACC tions were studied. Forty cases (80%) were in the first tri- TAC CAA CGT AGG TT-3Ј10 for the second round generated 234- mester and 10 cases (20%) were in the third trimester. (bp) and 168-bp products, respectively. There were 12 cases with normal pregnancy included as Amplification program was 94ЊC for 1 minute, 30 cycles (55ЊC a control group. The mean age Ϯ SD of the patients was for 1 minute, 72ЊC for 1 minute). Following PCR, the amplicon 26.8 Ϯ 5.61 years and the mean age of the control group Arch Pathol Lab Med—Vol 131, June 2007 High-Risk Pregnancies, Viral Infections, Screening—El-Sayed Zaki & Goda 957 Table 1. Serologic Results of Cytomegalovirus (CMV), Parvovirus, and Herpes Simplex Virus 1/Herpes Simplex Virus 2 (Herpes 1/2) Immunoglobulin M (IgM) Patients Control Virus No. % No. % P Value CMV IgM Positive 6 12 0 0 P ϭ .20 Negative 44 88 12 100 Parvovirus IgM Positive 42 84 2 20 P Ͻ .001 Negative 8 16 10 80 Herpes 1/2 IgM Positive 20 40 0 0 P ϭ .001 Negative 30 60 12 100 was 26.2 Ϯ 6.25 years with a statistically insignificant dif- ference between both groups (P ϭ .80). The range of gra- vidity between both groups was 2 to 7 times. Serologic Study for Parvovirus B19, HSV-1/HSV-2, and CMV Receiver operative curve for parvovirus immunoglobulin M. Diagonal In the study of serologic responses for the viruses in segments are produced by ties. RSA, parvovirus IgM had the highest rate (84%), followed by HSV-1/HSV-2 IgM (40%), and CMV IgM (12%). In the pregnant women group, only parvovirus IgM was positive mL with a sensitivity of 83.3%, specificity of 66.7%, and in 2 cases (16.7%). There was a statistically significant dif- accuracy of 89.2% as shown by the receiver operator char- ference between the patient group and the control group acteristic curve (Figure). in prevalence of parvovirus IgM (P Ͻ .001) and HSV-1/ The sensitivity of parvovirus IgM was 100%, specificity HSV-2 IgM (P ϭ .001) (Table 1). was 47.4%, accuracy was 67.7%, positive predictive value was 54.5%, and negative predictive value was 100%. The PCR Study for Parvovirus B19, HSV-1/HSV-2, and CMV sensitivity of HSV-2 IgM was 100%, specificity was 91.3%, In the PCR study for viral DNA, parvovirus B19 was positive predictive value was 80%, and negative predictive positive in 24 RSA (48%), HSV-2 was positive in 16 RSA value was 100% (Table 3). Ϯ (32%), and CMV was positive in 6 RSA (12%) patients. From positive parvovirus IgM cases, the mean SD of Ϯ There was a statistically significant difference between IgM value for PCR positive cases was 78.5 30.12 IU/ Ϯ RSA patients and pregnant women without RSA in par- mL, and for PCR negative cases the value was 30.02 vovirus PCR and HSV-2 PCR (P ϭ .002 and P ϭ .02, re- 17.64 IU/mL with a statistically significant difference be- Ͻ spectively) (Table 2). tween both levels (P .001) (Table 4). Value of the Serologic Markers Compared With PCR COMMENT When trying to detect a cutoff value for parvovirus B19 Cytomegalovirus and parvovirus are linked both to late 2,13 IgM with improved specificity, the best one was 48 IU/ abortions and to stillbirth. Infection with parvovirus B19 during pregnancy is known to be associated with var- ious fetal damage such as aplastic anemia and hydrops 14 Table 2. Polymerase Chain Reaction (PCR) Results of fetalis. Reactivation of chronic CMV infection in the DNA Detection for Cytomegalovirus (CMV), course of pregnancy might result in fetal infection with Parvovirus, and Herpes Simplex Virus 1/Herpes spontaneous abortion.15 Simplex Virus 2 (Herpes 1/2) In our study, the highest frequency of viral markers was Patients Control for parvovirus B19 followed by HSV-2 and CMV IgM in RSA patients. Lin et al16 reported that parvovirus IgM was Virus No. % No. % Difference detected in 36.4% of random female samples in . CMV PCR In Kuwait, seroprevalence of parvovirus IgG and IgM Positive 6 12 0 0 ␹2 ϭ 1.59, were 53.3% and 2.2%, respectively, in pregnant women P ϭ .20 without recurrent abortions with seroconversion rates of Negative 44 88 12 100 16.5%.14 In a Swedish study, the prevalence of parvovirus Parvovirus PCR was 81% with 6.8% seroconversion after labor.17 Positive 24 48 0 0 ␹2 ϭ 9.39, In Russia, the positivity of parvovirus B19 IgG was 66.9% P ϭ .002 and for CMV was 81.1% among pregnant aborters.18 Negative 26 52 12 100 The discrepancy between results of serologic tests in Herpes 1/2 PCR various studies for parvovirus B19 might be the result of Positive 16 32 0 0 ␹2 ϭ 5.17, the differences of the studied populations. In addition, the P ϭ .02 difference in gravidity might affect the rate of spread of Negative 34 68 12 100 parvovirus, which is an infectious disease transmitted 958 Arch Pathol Lab Med—Vol 131, June 2007 High-Risk Pregnancies, Viral Infections, Screening—El-Sayed Zaki & Goda Table 3. Values of Serological Markers for the Studied Viruses Compared With Polymerase Chain Reaction Positive Predictive Negative Predictive Virus* Sensitivity, % Specificity, % Accuracy, % Value, % Value, % CMV IgM 100 100 100 100 100 Parvovirus IgM 100 47.4 67.7 54.5 100 Herpes 2 IgM 100 91.3 93.5 80 100 * CMV indicates cytomegalovirus; IgM, immunoglobulin M; and Herpes 2, herpes simplex virus type 2. mainly by children.19 This also explains the presence of 2 PCR negative for virus, which led to a decrease in its spec- cases positive for parvovirus B19 in the control group. ificity. However, those cases might be recent infections However, the high detection rate of parvovirus among pa- with positive when DNA was no longer detected. tients with recurrent abortions supports the hypothesis On the other hand, DNA measurement is the best in- that parvovirus could be the leading cause of early recur- dicator of infection not only in the fetal blood but also in rent abortions. the maternal blood. This improves the diagnostic value of The lower rate of positive CMV IgM could be attributed the laboratory results considerably. DNA assays are essen- to the fact that primary CMV infection is usually acquired tial in cases of doubtful serologic results.25 during childhood, so the serosusceptibility and the risk of In a trial to improve the diagnostic value of parvovirus primary infection is lower during pregnancy than with IgM, we evaluated a cutoff value that diagnosed all par- other viruses.18 vovirus-positive RSA with PCR. The value was 78.5 Ϯ Herpes simples virus 1/2 IgM was positive in 80% of 30.12 IU/mL compared with 30.02 Ϯ 17.64 IU/mL in cas- women with recurrent abortions.20 In another study, se- es with PCR negative for parvovirus. There was a statis- rology to HSV was positive in 32% of pregnant women.21 tically significant difference (P Ͻ .001). This can be used It seems that the state of pregnancy predisposes to HSV as a simple approach to predict cases with parvovirus vi- reactivation, so pregnant females either with recurrent remia. abortions or with normal pregnancy display serologic Another approach was plotting the receiver operator markers of HSV reactivation.9 However, in our study there characteristic to find the value with improved specificity was a significant increase of HSV in patients with recur- of parvovirus IgM. The value was 48 IU/mL, with 83.3% rent abortions compared with pregnant patients without sensitivity, 66.7% specificity, and 89.2% accuracy. recurrent abortions, which denotes that HSV may predis- From this study, we conclude that viral infections with pose to this condition. parvovirus B19 and HSV-2 might play a role in recurrent The sensitivity of parvovirus IgM when compared with abortions and careful investigation for such conditions PCR was 100%, specificity was 47.4%, accuracy was 67.7%, must involve detecting the presence of these viruses. De- positive predictive value was 54.5%, and negative predic- tecting specific IgM to parvovirus and to HSV-2 also ap- tive value was 100%. In a study of 29 pregnant women pears to be a reliable screening indicator for pregnancies with after exclusion of fetomaternal incom- at risk. Polymerase chain reaction determination of viral patibility, 9 (31%) had active parvovirus infection with DNA in maternal serum improves the diagnostic value of positive DNA by PCR; only 3 of them had positive IgM.22 the serologic tests and leads us to conclude that both test A lower rate of positivity for parvovirus was detected by methods would be effective tools in determining the pos- Wermelinger et al,5 who found 16 of 212 cases with re- sibility of recurrent spontaneous abortion. current abortions. References The value of serologic determination of parvovirus IgM 1. Penta M, Lukic A, Conte MP. Infectious agents in tissue from spontaneous 23 was discussed by Gallinella et al who reported that IgM abortions in the first trimester of pregnancy. New Microbiol. 2003;26:329–337. determination detected only 60% of parvovirus B19–doc- 2. Lazzarotto T, Varani S, Spezzacatena P. Maternal IgG and IgM detected by umented infection. Immunoglobulin M was fundamental blot as diagnostic tools to identify pregnant women at risk of transmitting cyto- megalovirus. Viral Immunol. 2000;13:137–141. as it is the unique diagnostic marker in 20.8% of docu- 3. Levy R, Werssman A, Blomberg G. Infection by parvovirus B19 during preg- mented infections and in the diagnosis of recent symp- nancy: a review. Obstet Gynecol Surv. 1997;52:254–259. tomatic infection when B19 DNA was no longer detectable. 4. Corcoran A, Doyle S. Advances in the biology, diagnosis and host 24 interactions of parvovirus B19. J Med Microbiol. 2004;53:459–475. In addition, Hoebe et al reported contradictory results 5. Wermelinger MC, Delemann WM, Lina deMendonca MC. Detection of hu- between serologic results and PCR and demonstrated that man parvovirus B19 infection: a study of 212 suspected cases in the state of Rio maximum sensitivity of parvovirus testing would require de Janeiro, Brazil. J Clin Virol. 2002;25:223–230. 6. Zerbini M, Gallinella G, Cricca M. Diagnostic procedures in B19 infection. both tests to be performed. Pathol Biol (Paris). 2002;50:332–338. In our study, IgM correctly diagnosed all cases with 7. Cusini M, Ghislanzoni M. The importance of diagnosing genital herpes. J PCR positive for virus but diagnosed more cases with Antimicrob Chemother. 2001;47(19):16–20. 8. Bujko M, Sulovic V, Zivanovic V. Herpes simplex virus infection in women with previous spontaneous abortion. J Perinat Med. 1998;16(3):193–196. 9. Sifakis S, Koumantakis E, Koffa M. Detection of herpes simplex virus (HSV) Table 4. Parvovirus Immunoglobulin M (IgM) Value in aborted material using the polymerase chain reaction technique. Gynaecol Obstet Invest. 1998;45:109–115. for Polymerase Chain Reaction (PCR) Positive and 10. Priya K, Madhavan HN. Use of nested polymerase chain reaction (nPCR) Negative Results for the detection of cytomegalovirus (CMV) in clinical specimens. Indian J Med Parvovirus IgM Res. 2002;115:5–10. 11. Johnson G, Nelson S, Petric M, Tellier R. Comprehensive PCR-based assay Positive PCR Positive PCR Negative for detection and species identification of herpes viruses. J Clin Microbiol. Mean Ϯ SD 78.5 Ϯ 30.12 30.02 Ϯ 17.64 2000;38:3274–3279. t test 7.99 12. Clewley JP. Polymerase chain reaction assay of parvovirus B19 DNA in P Ͻ.001 clinical specimens. J Clin Microbiol. 1989;27:2647–2651. 13. Corcoran A, Mahone T, Parland P. Exvivo cytokine responses against par-

Arch Pathol Lab Med—Vol 131, June 2007 High-Risk Pregnancies, Viral Infections, Screening—El-Sayed Zaki & Goda 959 vovirus B19 antigens in previously infected women. J Med Virol. 2003;70:475– 20. Panum Jensen L, Thorsen P, Jeune B. An epidemic of parvovirus B 19 in a 480. population of 354 pregnant women: a study of sociodemographic and medical 14. Maksheed M, Pacsa AS, Essa SS. The prevalence of antibody to human risk factors. Br J Obstet Gynaecol. 2000;107:637–643. parvovirus B19 in pregnant women in Kuwait. Acta Trop. 1999;15:225–229. 21. Frenkel LM, Garratty EM, Shen JP. Clinical reactivation of herpes simplex 15. Szkaradkiewicz A, Pieta P, Tulecka T. The diagnostic value of anti-CMV virus type 2 infection in seropositive pregnant women with no history of genital and anti HPV, B19 antiviral in studies on causes of recurrent abortions. herpes. Ann Intern Med. 1993;118:414–418. Ginekol Pol. 1997;68(4):181–186. 22. Lenkiewicz B, Roszkowski T, Grabarczyk P. Diagnosis of human parvovirus 16. Lin KH, You SL, Chen CJ. Sero epidemiology of human parvovirus B19 in B19 infection in non immune hydropes fetalis. Ginekol Pol. 1998;69(4):175–181. Taiwan. J Med Virol. 1999;57:169–173. 23. Gallinella G, Zuffi E, Gentilomi G. Relevance of B19 markers in serum 17. Skjoldebrandsparre L, Tofventamt, Papadogiannakis N. Parvovirus B19 in- samples for a diagnosis of parvovirus B19 correlated diseases. J Med Virol. 2003; fection: association with third trimester intrauterine fetal death. Br J Obstet Gy- 71:135–139. naecol. 2000;107:476–480. 18. Odland J, Sergejeva IV, Ivaneev MD. Seropositivity of cytomegalovirus, 24. Hoebe CJ, Claas EC, Steenberg JE. Confirmation of an outbreak of parvo- parvovirus and in pregnant women and recurrent aborters in Leningrad virus B19 in a primary school using IgM ELISA and PCR on thumb prick blood country, Russia. Acta Obstetricia Gynecol Scand. 2001;80:1025–1030. samples. J Clin Virol. 2002;25:303–307. 19. Huang LI, Zhang Z. The relationship between herpes simplex virus 11, 25. Dieck D, Scbild RL, Hansman M. Prenatal diagnosis of congenital parvo- human papilloma virus infection and infertility after artificial abortion. Zhonghua virus B19 infection: value of serological and PCR techniques in maternal and Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 1998;12:155–157. fetal serum. Prenatal Diag. 1999;19(12):119–123.

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