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Relevance of Parvovirus B19, Herpes Simplex Virus 2, and Cytomegalovirus Virologic Markers in Maternal Serum for Diagnosis of Unexplained Recurrent Abortions

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Relevance of Parvovirus B19, Herpes Simplex Virus 2, and Cytomegalovirus Virologic Markers in Maternal Serum for Diagnosis of Unexplained Recurrent Abortions Relevance of Parvovirus B19, Herpes Simplex Virus 2, and Cytomegalovirus Virologic Markers in Maternal Serum for Diagnosis of Unexplained Recurrent Abortions Maysaa El-Sayed Zaki, MD; Hossam Goda, MD ● Context.—The impact of viral infections 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, herpes simplex virus 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 viruses compared mean ؎ SD of IgM value was 78.5 ؎ 30.12 IU/mL, and ؎ with their genomes 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 stillbirth.4,5 loss, but the implication and prevalence of microorgan- Because B19 infection 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 anemia or rash can be com- lished.1 mon to other pathogens, 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 genome in clinical samples.6 Another virus that could be implicated in recurrent abortion is herpes simplex. Genital herpes 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 transmission 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.
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