Laboratory Assessment and Diagnosis of Congenital Viral Infections

Home , Herpes simplex virus, Neonatal herpes simplex, Rubella virus

Reproductive Toxicology 21 (2006) 350–382

Review Laboratory assessment and diagnosis of congenital viral infections: Rubella, cytomegalovirus (CMV), varicella-zoster virus (VZV), herpes simplex virus (HSV), parvovirus B19 and human immunodeﬁciency virus (HIV) Ella Mendelson a,∗, Yair Aboudy b, Zahava Smetana c, Michal Tepperberg d, Zahava Grossman e a Central Virology Laboratory, Ministry of Health and Faculty of Life Sciences, Bar-Ilan University, Chaim Sheba Medical Center, Tel-Hashomer, 52621, Israel b National Rubella, Measles and Mumps Center, Central Virology, Laboratory, Ministry of Health, Chaim Sheba Medical Center, Tel-Hashomer, Israel c National Herpesvirus Center, Central Virology Laboratory, Ministry of Health, Chaim Sheba Medical Center, Tel-Hashomer, Israel d CMV Reference Laboratory, Central Virology Laboratory, Ministry of Health, Chaim Sheba Medical Center, Tel-Hashomer, Israel e National HIV, EBV and Parvovirus B19 Reference Laboratory, Central Virology Laboratory, Ministry of Health, Chaim Sheba Medical Center, Tel-Hashomer, Israel Received 14 October 2004; received in revised form 30 January 2006; accepted 7 February 2006

Abstract Viral infections during pregnancy may cause fetal or neonatal damage. Clinical intervention, which is required for certain viral infections, relies on laboratory tests performed during pregnancy and at the neonatal stage. This review describes traditional and advanced laboratory approaches and testing methods used for assessment of the six most signiﬁcant viral infections during pregnancy: rubella virus (RV), cytomegalovirus (CMV), varicella-zoster virus (VZV), herpes simplex virus (HSV), parvovirus B19 and human immunodeﬁciency virus (HIV). Interpretation of the laboratory tests results according to studies published in recent years is discussed. © 2006 Elsevier Inc. All rights reserved.

Keywords: Laboratory diagnosis; Congenital viral infections; Rubella; Cytomegalovirus (CMV); Varicella-zoster virus (VZV); Herpes simplex virus (HSV); Parvovirus B19; Human immunodeﬁciency virus (HIV)

Contents

1. General introduction...... 352 2. Rubella virus ...... 352 2.1. Introduction ...... 352 2.1.1. The pathogen ...... 352 2.1.2. Immunity and protection...... 352 2.1.3. Laboratory assessment of primary rubella infection in pregnancy ...... 355 2.1.4. Pre- and postnatal laboratory assessment of congenital rubella infection ...... 357 2.2. Laboratory assays for assessment of rubella infection and immunity...... 358 2.2.1. Rubella neutralization test (NT) ...... 358 2.2.2. Hemagglutination inhibition test (HI) ...... 358 2.2.3. Rubella specific ELISA IgG...... 358 2.2.4. Rubella specific ELISA IgM ...... 358 2.2.5. Rubella specific IgG-avidity assay ...... 359 2.2.6. Rubella virus isolation in tissue culture ...... 359

∗ Corresponding author. Tel.: +972 3 530 2421; fax: +972 3 535 0436. E-mail address: [email protected] (E. Mendelson).

2.2.7. Rubella RT-PCR assay ...... 359 2.3. Summary ...... 359 3. Cytomegalovirus (CMV)...... 360 3.1. Introduction ...... 360 3.1.1. The pathogen ...... 360 3.1.2. Laboratory assessment of CMV infection in pregnant women...... 360 3.1.3. Prenatal assessment of congenital CMV infection ...... 360 3.2. Laboratory assays for assessment of CMV infection ...... 361 3.2.1. CMV IgM assays ...... 361 3.2.2. CMV IgG assays...... 361 3.2.3. CMV IgG avidity assays ...... 361 3.2.4. CMV neutralization assays...... 362 3.2.5. Virus isolation in tissue culture ...... 362 3.2.6. Detection of CMV by PCR ...... 362 3.2.7. Quantitative PCR-based assays ...... 362 3.3. Summary ...... 363 4. Varicella-zoster virus (VZV) ...... 363 4.1. Introduction ...... 363 4.1.1. The pathogen ...... 363 4.1.2. Assessment of VZV infection in pregnancy ...... 363 4.1.3. Prenatal and perinatal laboratory assessment of congenital VZV infection ...... 364 4.2. Laboratory assays for assessment of VZV infection and immunity ...... 364 4.2.1. VZV IgG assays ...... 364 4.2.2. VZV IgM assays ...... 364 4.2.3. Virus detection in clinical specimens ...... 364 4.2.4. Virus isolation in tissue culture ...... 364 4.2.5. Direct detection of VZV antigen ...... 364 4.2.6. Molecular methods for detection of viral DNA ...... 364 4.3. Summary ...... 365 5. Herpes simplex virus (HSV)...... 365 5.1. Introduction ...... 365 5.1.1. The pathogen ...... 365 5.1.2. Laboratory assessment of HSV infection in pregnancy and in neonates ...... 365 5.2. Laboratory assays for assessment of HSV infection and immune status ...... 366 5.2.1. HSV IgG assays ...... 366 5.2.2. HSV type-speciﬁc IgG assays ...... 366 5.2.3. HSV IgM assays ...... 366 5.2.4. Virus isolation in tissue culture ...... 366 5.2.5. Direct antigen detection of HSV ...... 366 5.2.6. Detection of HSV DNA by PCR ...... 366 5.3. Summary ...... 367 6. Parvovirus B19 ...... 367 6.1. Introduction ...... 367 6.1.1. The pathogen ...... 367 6.1.2. Laboratory assessment of parvovirus B19 infection in pregnancy ...... 367 6.1.3. Prenatal laboratory assessment of congenital B19 infection ...... 367 6.2. Laboratory assays for assessment of parvovirus B19 infection ...... 368 6.2.1. B19 IgM and IgG assays...... 368 6.2.2. Detection of viral DNA in maternal and fetal specimens ...... 368 6.2.3. Quantitative assays for detection of viral DNA ...... 369 6.3. Summary ...... 369 7. Human immunodeﬁciency virus ...... 369 7.1. Introduction ...... 369 7.1.1. The pathogen ...... 369 7.1.2. Importance of laboratory assessment of HIV infection in pregnancy ...... 370 7.1.3. Prenatal laboratory assessment of HIV infection ...... 370 7.2. Laboratory assessment of HIV infection ...... 370 7.2.1. HIV antibody assays ...... 370 7.2.2. Detection of viral DNA in maternal and newborn specimens...... 371 7.3. Summary ...... 372 Acknowledgments ...... 372 References ...... 372 352 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382

1. General introduction Since the algorithm for maternal and fetal assessment and the interpretation of tests results vary from one virus to another, we Viral infections during pregnancy carry a risk for intrauterine have described the approach to each viral infection in a separate transmission which may result in fetal damage. The conse- chapter. Figs. 1–6 depict the most common algorithms used for quences of fetal infection depend on the virus type: for many the laboratory diagnosis of each of the viral infections common viral infections there is no risk for fetal damage, but some viruses are teratogenic while others cause fetal or neonatal 2. Rubella virus diseases ranging in severity from mild and transient symptoms to a fatal disease. In cases where infection during pregnancy 2.1. Introduction prompts clinical decisions, laboratory diagnostic tests are an essential part of the clinical assessment process. This review 2.1.1. The pathogen describes the six most important viruses for which laboratory Rubella is a highly transmissible childhood disease which assessement during pregnancy is required and experience has can cause large outbreaks every few years. It is a vaccine pre- been gained over many years. Rubella virus and CMV are ter- ventable disease and in developed countries outbreaks are mostly atogenic viruses, while VZV, HSV, parvovirus B19 and HIV confined to unvaccinated communities [1]. Rubella reinfection cause fetal or neonatal transient or chronic disease. following natural infection is very rare. Rubella virus (RV) is The ability of viruses to cross the placenta, infect the fetus classified as a member of the togaviridae family and is the only and cause damage depends, among other factors, on the mother’s virus of the genus rubivirus [2]. Hemagglutinating activity and at immune status against the specific virus. In general, primary least three antibody neutralization domains were assigned to the infections during pregnancy are substantially more damaging early proteins E1 and E2 [3–5]. At least one weak neutralization than secondary infections or reactivations. domain was identified on E2 [5]. Laboratory testing of maternal immune status is required to The main route of postnatal virus transmission is by direct diagnose infection and distinguish between primary and sec- contact with nasopharyngial secretions [6]. Postnatal RV infec- ondary infections. Assessment of fetal damage and prognosis tion is a generally mild and self-limited illness [6–8], but primary requires prenatal laboratory testing primarily in those cases RV infections during the first trimester of pregnancy have high where a clinical decision such as drug treatment, pregnancy ter- teratogenic potential leading to severe consequences, known as mination or intrauterine IgG transfusion must be taken. congenital rubella syndrome (CRS) which may occur in 80–85% This review describes basic virological facts and explains of cases [8,9]. It should be emphasized that more than 50% of RV the laboratory approaches and techniques used for the diagnos- infections in non-immunized persons in the general population tic process. It aims at familiarizing physicians with the rational (and in pregnant women) are subclinical [6–9]. behind the laboratory requests for specific and timely specimens and with the interpretation of the tests results including its lim- 2.1.2. Immunity and protection itations. Antibody level of 10–15 international units (IU) of IgG per The laboratory methods used for assessment of viral infec- millilitre is considered protective. Naturally acquired rubella tions in general are of two categories: serology and virus generally confers lifelong and usually high degree of immu- detection. Serology is very sensitive but often cannot conclu- nity against the disease for the majority of individuals [10,11]. sively determine the time of infection, which may be critical Rubella vaccination induces immunity that confers protection for risk assessment. Traditional serological tests, which mea- from viraemia in the vast majority of vaccinees, which usually sure antibody levels without distinction between IgM and IgG, persists for more than 16 years [10–12]. A small fraction of the usually require two samples for determination of seroconver- vaccinees fail to respond or develop low levels of detectable sion or a substantial rise in titer. The modern tests can dis- antibodies which may decline to undetectable levels within 5–8 tinguish between IgG and IgM and may allow diagnosis in years from vaccination [13–17]. one serum sample. However, biological and technical difficul- Several methods are used to determine immunity (Table 1). ties are common and may cause false positive and false neg- Neutralization test (NT) and hemagglutination inhibition test ative results. The properties of all serological assays used for (HI) correlate well with protective immunity, but since they are each of the viruses will be described in detail in the following difficult to perform and to standardize, they were replaced by chapters. the more rapid, facile and sensitive enzyme-linked immunosor- Virus detection is used primarily for prenatal diagnosis. Inva- bant assay (ELISA) [5,18]. In our experience (unpublished data), sive procedures must be used to obtain samples representing the there is a clear distinction between antibody levels measured fetus such as amniotic fluid (AF), cord blood and chorionic villi using ELISA, and antibody levels measured using functional (CV). The traditional “gold standard” assay for virus detection assays such as NT and HI. Moreover, standardization of anti used to be virus isolation in tissue culture, but other, more rapid RV antibody assays using different techniques and a variety and sensitive methods were developed in recent years. Among of antigens (i.e., whole virus, synthetic peptides, recombinant the new methods are direct antigen detection by specific anti- antigen, etc.) has not been achieved, leading to uncertainties bodies and amplification and detection of viral nucleic acids. regarding the antibody levels that confer immunity and protec- The general characteristics of all laboratory assays described in tion against reinfections and against virus transmission to the this article are summarized in Table 1. fetus [19–26]. Most of the reinfection cases (9 out of 18 cases; E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 353

Table 1 Summary and characteristics of the laboratory tests used for assessment of viral infections in pregnancy Laboratory test Test principles Clinical Technical advantages Technical limitations Interpretation of samples disadvantages positive results

Serology Neutralization (NT) Inhibition of virus Maternal Corresponds with Laborious, not Done only in Neutralizing growth in tissue blood protection very sensitive, not reference laboratories antibodies are present culture by Aba Ab class-specific at a certain titer Hemagglutination Prevention of Maternal Accurate and Laborious, not Ab Used only for rubella, HI antibodies are inhibition (HI) hemagglutination by blood corresponds with class specific done only in present at a certain binding of Ab to protection specialized labs titer viral Agb ELISA IgM Detection of virus Maternal Fast and sensitive, None False positive and IgM antibodies are specific Ab bound to blood, fetal commercialized, false negative present a solid phase by a blood, automated labled secondary newborn blood anti-IgM Ab ELISA IgG Detection of virus Maternal Fast and sensitive, None None IgG antibodies are specific Ab bound to blood, commercialized, present (sometimes a solid phase by a newborn blood automated with units) labled secondary anti-IgG Ab IgG avidity Removal of low Maternal Fast and sensitive, Not many No interpretation for Low avidity: recent (ELISA) avidity IgG Ab blood commercialized, available results outside the infection; medium which results in a automated commercially inclusion or exclusion avidity: not known; reduced signal criteria high avidity: probably old infection Immunofluores- Detection of IgG or Maternal Can yield titer; short Manual, reading is Unsuitable for testing Antibodies are present cence (IFA; IgM Ab which binds blood, fetal time subjective large numbers at a certain titer IFAMA, etc.) to a spot of virus blood, infected cells on a newborn blood slide by a labled secondary Ab Western blot (WB) Separated viral Maternal Detects antibody Laborious Not very sensitive Antibodies specific to proteins attached to blood infant’s specific to a viral certain viral antigens a nylon membrane blood protein are present react with patient’s serum and detected by labled anti-human Ab Virus detection Virus isolation in Innoculation of Any clinical Detects and isolates Very labourious Insensitive, done only Live virus is present tissue culture specific tissue sample which live virus Slow in virology labs in the clinical sample cultures with may contain clinical samples and virus watching for CPEc Direct antigen Detection of a viral For IFA: cells Fast and simple Not sensitive Not sensitive, low The sample most detection antigen in cells from from clinical positive predictive likely contains live a clinical sample by samples. For value virus IFA or ELISA ELISA: any sample Shell-vial assay Innoculation of Any clinical Detects live virus; Labourious; Not highly sensitive; Live virus is present specific tissue sample which rapid: results within requires high done only in virology in the clinical sample cultures with may contain 16–72 h skills; uses labs clinical samples, virus expensive then fixation and monoclonal Abs detection of viral cell-bound antigen by IFA Molecular assays PCR; RT-PCR Enzymatic Any clinical Fast, simple, can be Very prone to False positive by Viral nucleic acid is amplification of sample which automated; very contaminations contamination; may present in the sample, viral nucleic acid may contain sensitive detect latent virus not known if live virus and detection of virus is present amplified sequences 354 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382

Table 1 (Continued ) Laboratory test Test principles Clinical Technical advantages Technical limitations Interpretation of samples disadvantages positive results

Real-time Detection of Any clinical Very fast, simple not Expensive Sometimes too Viral nucleic acid is PCR/RT-PCR accumulating PCR sample which prone to instruments sensitive, present in the sample products by a may contain contaminations; can interpretation of very (at a certain amount), fluorescent dye or virus be quantitative low result not known if live virus probe in a questionable is present specialized instrument In situ Detection of viral Cells or tissue Sensitive and specific Difficult to Done only in Viral nucleic acid is hybridization nucleic acid in from clinical perform specializing labs present in the sample, smears or tissue samples not known if live virus sections by labled is present probes In situ PCR Detection of viral Cells or tissue Sensitive and specific Difficult to Doe only in Viral nucleic acid is nucleic acid in from clinical perform specializing labs present in the sample, smears or tissue samples not known if live virus sections by PCR is present using labled primers a Antibody. b Antigen. c Cytopathic effect.

50%) which were detected during an outbreak in Israel in 1992 considerably higher than following natural infection, ranging occurred in the presence of low neutralizing antibody titers of between 10% and 20% [19]. 1:4 (cut off level), and sharp decline in the reinfection rate corre- Many developed countries adopted the infant routine vacci- lated with the presence of higher titers of neutralizing antibodies nation policy using MMR (mumps measles and rubella) vaccine (unpublished data). Reinfection rates following vaccination are designed to provide indirect protection of child-bearing age

Fig. 1. Algorithm for assessment of rubella infection in pregnancy: the algorithm shows a stepwise procedure beginning with testing of the maternal blood for IgM and IgG. If the maternal blood is IgM negative the IgG result determines if the woman is seropositive (immune) or seronegative (not immune). If not immune the woman should be retested monthly for seroconversion till the end of the 5th month of pregnancy. If the maternal blood is IgM and IgG positive the next step would be an IgG avidity assay on the same blood sample to estimate the time of infection. Low avidity index (AI) indicates recent infection while high AI indicates past or recurrent infection. Medium AI is inconclusive and the test should be repeated on a second blood sample obtained 2–3 weeks later. If the maternal blood is IgM positive and IgG negative, recent primary infection is suspected and the same tests should be repeated on a second blood sample obtained 2–3 weeks later. If the results remain the same (IgM+ IgG−), then the IgM result is considered non-speciﬁc, indicating that the woman has not been infected (however she is seronegative and should be followed to the end of the 5th month as stated above). If the woman has seroconverted (IgM+ IgG+), recent primary infection is conﬁrmed and prenatal diagnosis should take place if the woman wishes to continue her pregnancy. Determination of IgM in cord blood is the preferred method with the highest prognostic value. Post natal diagnosis is based on the newborn’s serology (IgM for 6–12 m and IgG beyond age 6 m) and on virus isolation from the newborn’s respiratory secretions. E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 355

Fig. 2. Algorithm for assessment of CMV infection in pregnancy: the algorithm shows a stepwise procedure which begins with detection of IgM in maternal blood. If the maternal blood is IgG positive, an IgG avidity assay on the same blood sample should be performed to estimate the time of infection. Low avidity index (AI) indicates recent primary infection and prenatal diagnosis should follow. Medium or high AI is mostly inconclusive, especially if the maternal blood was obtained on the second or third tremester. Continuation of the assessment is based on either maternal blood or fetal prenatal diagnosis. If the first maternal blood was IgM positive but IgG negative, a second blood sample should be obtained 2–3 weeks later. If the IgG remains negative then the IgM is considered non-specific. If the woman has seroconverted and developed IgG, primary infection is confirmed and prenatal diagnosis should follow. For prenatal diagnosis amniotic fluid (AF) should be obtained not earlier than the 21st week of gestation and 6 weeks following seroconversion. Fetal infection is assessed by virus isolation using standard tissue culture or shell-vial assay, and/or by PCR detection of CMV DNA. Positive result by either one of these tests indicates fetal infection. women regardless of vaccination status. However, in Israel and an increase in the number of therapeutic abortions and reducing in other countries with high vaccination coverage, RV still cir- the number of CRS cases. However, frequently the low level culates and may cause reinfections in vaccinated women whose of IgM detected is not indicative of a recent primary infec- immunity has waned [19,20,23, unpublished data]. tion for several reasons: (a) IgM reactivity after vaccination or primary rubella infection may sometimes persist for up to 2.1.3. Laboratory assessment of primary rubella infection several years [27–29]; (b) heterotypic IgM antibody reactivity in pregnancy may occur in patients recently infected with Epstein Barr virus Assessment of primary rubella infection in pregnant women (EBV), cytomegalovirus (CMV), human parvovirus B19 and relies primarily on the detection of specific maternal IgM anti- other pathogens, leading to false positive rubella IgM results bodies in combination with either seroconversion or a >4-fold [30–35]; (c) false positive rubella specific IgM response may rise in rubella specific IgG antibody titer in paired serum sam- occur in patients with autoimmune diseases such as systemic ples (acute/convalescent) as shown in Fig. 1. Today, due to lupus erythematosus (SLE) or juvenile rheumatoid arthritis, etc., the high sensitivity of the ELISA-IgM assays low levels of due to the presence of rheumatoid factor (RF) [36,37]; (d) low rubella specific IgM are detected more frequently, leading to level of specific rubella IgM may occur in pregnancy due to

Fig. 3. Algorithm for assessment of VZV infection in pregnancy: two situations are shown: (1) clinical varicella in a pregnant woman (top left) should be assessed by serology (IgM and IgG in maternal blood) and by virus isolation or detection in early dermal lesions. If either of those approaches conﬁrms maternal VZV infection (positive virus isolation/detection test and/or maternal seroconversion), then fetal infection can be assessed by virus detection in amniotic ﬂuid using direct antigen detection or PCR. (2) Exposure of a pregnant woman to a varicella case (top right) should prompt maternal IgG testing within 96 h from exposure. If the mother has no IgG (not immune) she should receive VZIG within 96 h from exposure. 356 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382

Fig. 4. Algorithm for assessment of HSV infection in pregnancy and in neonates: the algorithm shows two complementary approaches to the confirmation of genital HSV infection in pregnant women. (1) If genital lesions are present (top right), virus isolation and typing is the preferred diagnostic approach. A positive woman should be examined during delivery for genital lesions. If normal delivery has taken place, the newborn should be examined for HSV infection symptoms and tested by virus isolation or PCR using swabs taken from skin, eye, nasopharynx and rectum or CSF. Detection of IgM in the newborn’s blood also confirms the diagnosis. Negative infants should be followed for 6 month. (2) Serology (top left) is a stepwise procedure beginning with maternal IgM and IgG testing. The interpretation of the results is shown: low positive or negative IgM in the presence of IgG indicates previous infection with the same virus type (reactivation), or recurrent infection with the other virus type. Type-specific serology may resolve the issue. If the IgG test is negative, in the presence or absence of IgM, a second serum sample should be obtained to observe seroconversion. If the IgG remains negative then no infection occurred. If the woman seroconverted, type specific serology can identify the infecting virus type.

Fig. 5. Algorithm for assessment of parvovirus B19 infection in pregnancy: the algorithm shows a stepwise procedure beginning with maternal serology following clinical symptoms in the mother or in the fetus or maternal contact with a clinical case. Negative IgM and positive IgG indicate past infection, but if the IgG is high recent infection cannot be ruled out. In all other cases a second serum sample should be obtained and retested. Only in the case of repeated negative results for both IgG and IgM recent infection with B19 can be ruled out. In all other cases the fetus should be observed for clinical symptoms and if present tested for B19 infection by nested PCR or rt-PCR performed on amniotic ﬂuid or fetal blood. Positive result conﬁrms fetal infection while negative result suggests that the fetus was not infected with B19. E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 357

Fig. 6. Algorithm for assessment of HIV infection in pregnancy and in newborns to infected mothers: (1) diagnosis of maternal infection (top left) is by the routine protocol (testing first by EIA and confirming by WB or IFA). If the mother is positive she should be treated as described in the text. (2) A newborn to an HIV positive mother (top right) should be tested at birth by DNA PCR on a blood sample. The results, whether positive or negative, should be confirmed by retesting either immediately (if positive) or 14–60 days later (if negative). If positive the newborn should be treated while if negative testing should be repeated at 3–6 months and again at 6–12 months. As a general rule, any PCR positive test in a newborn should be repeated on two different blood samples. After 12 months serological assessment can replace the PCR test as the infant has lost its maternal antibodies.

polyclonal B-cells activation trigerred by other viral infections chorionic villi (CV) samples or amniotic fluid (AF) specimens. [33,35,38]. The laboratory methods used for virus detection are virus iso- False negative results may also occur in samples taken too lation in tissue culture or amplification of viral nucleic acids by early during the course of primary infection. Thus, the presence RT/PCR (Table 1). However, using those methods for detection or absence of rubella specific IgM in an asymptomatic patient of rubella virus in AF and CV might be unreliable, particularly should be interpreted in accordance with other clinical and epi- in AF samples due to low viral load. Studies showed that rubella demiological information available and prenatal diagnosis may virus may be present in the placenta but not in the fetus, or it be required. can be present in the fetus but not in the placenta, leading to A novel assay developed recently to support maternal diag- false negative results [6,43,44]. Thus, according to one opin- nosis is the IgG avidity assay (Table 1) which can differentiate ion, detection of rubella virus in AF or CV does not justify the between antibodies with high or low avidity (or affinity) to the risk of fetal loss following these invasive procedures [45], while antigen. It is used when the mother has both IgM and IgG in the according to another opinion, laboratory diagnosis of fetal infec- first serum collected (Fig. 1). Following postnatal primary infection should combine a serological assay (detection of rubella tion with rubella virus, the specific IgG avidity is initially low specific IgM) with a molecular method (viral RNA detection) in and matures slowly over weeks and months [39–41]. Rubella order to enhance the reliability of the diagnosis [46]. A recent specific IgG avidity measurement proved to be a useful tool study showed 83–95% sensitivity and 100% specifity for detec- for the differentiation between recent primary rubella (clinical tion of RV in AF by RT/PCR [47]. and especially subclinical infection), reinfection, remote rubella Postnatal diagnosis of congenital rubella infection [9,27,36] infection or persistent IgM reactivity. This distinction is crit- is based on one or more of the following: ical for the clinical management of the case, since infection prompts a therapeutic abortion, reinfection requires fetal assess- a. Isolation of rubella virus from the infant’s respiratory secre- ment, while remote infection or non-specific IgM reactivity carry tions. no risk to the fetus [39–42]. b. Demonstration of rubella specific IgM (or IgA) antibodies in cord blood or in neonatal serum, which remain detectable for 2.1.4. Pre- and postnatal laboratory assessment of 6–12 months of age. congenital rubella infection c. Persistence of anti-rubella IgG antibodies in the infant’s Maternal primary infection prompts testing for fetal infection serum beyond 3–6 months of age. (Fig. 1). The preferred laboratory method for prenatal diagnosis is determination of IgM antibodies in fetal blood obtained by The principles, advantages and disadvantages of each labo- cordocentesis [27,43]. Other options include virus detection in ratory test, are described below. 358 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382

2.2. Laboratory assays for assessment of rubella infection 2.2.3. Rubella specific ELISA IgG and immunity The ELISA technique was established for detection of an increasing range of antibodies to viral antigens. In 1976, Voller 2.2.1. Rubella neutralization test (NT) et al. [58] developed an indirect assay for the detection of anti- Virus neutralization is defined as the loss of infectivity due to viral antibodies. The technique has been successfully applied reaction of a virus with specific antibody. Neutralization can be for the detection of rubella specific antibodies. used to identify virus isolates or, as in the case of rubella diagno- Almost all commercially available ELISA kits for the detec- sis, to measure the immune response to the virus [24,36,48].As tion of rubella specific IgG are of the indirect type, employing a functional test, neutralization has proven to be highly sensitive, rubella antigen attached to a solid phase (microtiter polystyrene specific and reliable technique, but it can be performed only in plates or plastic beads). The source of the antigen (peptide, virology laboratories which comprise only a small fraction of recombinant or whole virus antigen) affects the sensitivity and the laboratories performing rubella serology. specificity of the assay. After washing and removal of unbound Rubella virus produces characteristic damage (cytopathic antigen, diluted test serum is added and incubated with the effect, CPE) in the RK-13 cell line that was found most sen- immobilized antigen. The rubella specific antibodies present in sitive and suitable for use in rubella neutralization test. Other the serum bind to the antigen. Then, unbound antibodies are cells such as Vero and SIRC lines can be used if conditions removed by washing and an enzyme conjugated anti-human IgG are carefully controlled [36]. Principally, 2-fold dilutions of is added and further incubation is carried out. The quantity of the each test serum are mixed and incubated with 100 infectious conjugate that binds to each well is proportional to the concen- units of rubella virus under appropriate conditions. Then cell tration of the rubella specific antibodies present in the patient’s monolayers are inoculated with each mixture and followed for serum. The plates are then washed and substrate is added result- CPE. Control sera possessing known high and low neutralizing ing in color development. The enzymatic reaction is stopped antibody levels and titrations of the virus are included in each after a short incubation period, and optical density (OD) is mea- test run. The neutralization titer is taken as the reciprocal of sured by an ELISA-reader instrument. The test principle allows the highest serum dilution showing complete inhibition of CPE the detection of IgM as well by using an appropriate anti-human [25,36]. IgM conjugate [53,57]. In most commercial ELISA IgG assays the results are auto- 2.2.2. Hemagglutination inhibition test (HI) matically calculated and expressed quantitatively in interna- Until recently, assessment of rubella immunity and diagno- tional units (IU). When performed manually, the procedure takes sis of rubella infection has been carried out mainly by the HI approximately 3 h but automation has reduced it to about 30 min test which is based on the ability of rubella virus to agglutinate [57–59]. It is important to note that in order to obtain reliable red blood cells [49]. HI test is labor intensive, and is currently results, determination of a significant change in specific IgG performed mainly by reference laboratories. HI is the “gold activity in paired serum samples should always be performed in standard” test against which almost all other rubella screening the same test run and in the same test dilution. and diagnostic tests are measured. During the test, the agglu- The correlation between the ELISA and HI or NT titers tination is inhibited by binding of specific antibodies to the is not always high. This may be explained by the fact that viral agglutinin. Titers are expressed as the highest dilution the three methods detect antibodies directed to different anti- inhibiting hemagglutination under standardized testing condi- genic determinants [54]. Certain individuals fail to develop tions [50–53]. antibodies directed to protective epitopes such as the neutral- The HI antibodies increase rapidly after RV infection since izing domains of E1 and E2 due to a defect in their rubella the test detects both, IgG and IgM class-specific antibodies. specific immune responses [21] but they do develop antibod- A titer of l:8 is commonly considered negative (cut off level: ies directed to antigenic sub-regions of rubella virus proteins. 1:16) and a titer of ≥1:32 indicates an earlier RV infection or ELISA assays utilizing whole virus as antigen may fail to dis- successful vaccination and immunity. Seroconversion is inter- tinguish between these different antibody specificities. Thus, preted as primary rubella infection, and a 4-fold increase in titer seroconversion determined by ELISA based on a whole virus between two serum samples (paired sera) in the same test series, antigen does not necessarily correlate with protection against is interpreted as a recent primary rubella infection or reinfec- infection [52]. tion [52]. Considerable experience has been accumulated over the years in the interpretation of the clinical significance of HI 2.2.4. Rubella specific ELISA IgM titers [52–54], and the test results accurately correlate with clin- Commercially available ELISA kits for the detection of IgM ical protection [5,18]. Although HI is generally considered as are mainly of two types: not sensitive enough, in certain situations it is still in use for resolution of diagnostic uncertainties. a. Indirect ELISA: The principle of the assay was described Detection of rubella specific IgM class antibodies by HI above for rubella IgG except for using enzyme labeled anti- test which requires tedious methods for purification of IgM or human IgM as a conjugate. In this assay, false negative results removal of IgG [55,56], are no longer in use due to the develop- may occur due to a competition in the assay between specific ment of a variety of rapid, easy to perform and sensitive methods, IgG antibodies with high affinity (interfering IgG) while the of which ELISA is the most vastly used [18,57]. specific IgM have lower affinity for the antigen [31,32].Inthe E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 359

new generation ELISA assays this is avoided by the addition Rubella virus can be grown in a variety of primary cells and of an absorbent reagent for the removal of IgG from the test cell lines [36,65], but RK-13 and Verocell lines are the most sen- serum. False positive results may occur if rheumatoid factor sitive and suitable for routine use. In these cell systems rubella (RF: IgM anti-IgG antibodies) is present along with specific virus produces characteristic CPE. Since the CPE is not always IgG in the test serum. Absorption or removal of RF and/or clear upon primary isolation, at least two successive subpas- IgG is necessary prior to the assay to avoid such reactions sages are required [66]. When CPE is evident the identity of the [30–32,60]. virus isolates should be confirmed using immunological or other b. IgM capture ELISA: In these assays anti-human IgM anti- methods [36,65,67]. body is attached to the solid phase for capture of serum IgM. Rubella virus antigen conjugated to enzyme-labeled 2.2.7. Rubella RT-PCR assay anti-rubella virus antibody is added for detection. This type Reverse transcription followed by PCR amplification (RT- of assay eliminates the need for sample pretreatment prior PCR) is a rapid, sensitive and specific technique for detection to the assay [32,61]. As for the rubella virus antigens, most of rubella virus RNA in clinical samples using primers from assays are based on whole virus extracts, but recent develop- the envelope glycoprotein E1 open reading frame [45,46,68,69]. ments led to production of recombinant and synthetic rubella Coding sequences for a major group of antigenic determinants virus proteins [5,62]. are located between nucleotides 731 and 854 of the E1 gene of RV strain M33. This region is highly conserved in various 2.2.5. Rubella specific IgG-avidity assay wild type strains and is likely to be present in most clinical This assay is based on the ELISA IgG technique and samples from rubella infected patients. Specific oligonucleotide applies the elution principle in which protein denaturant, mostly primers located in this region were designed for amplification urea (but also diethylamine, ammonium thiocyanate, guanidine by RT-PCR [70–72]. Following rubella genomic RNA extraction hydrochloride, etc.) is added after binding of the patient’s serum. from clinical specimens and RT-PCR amplification, the product The denaturant disrupts hydrophobic bonds between antibody is visualized by gel electrophoresis. Positive samples show a and antigen, and thus, low avidity IgG antibodies produced dur- specific band of the expected size compared to size markers ing the early stage of infection are removed. This results in a [68,69,72]. significant reduction in the IgG absorbance level [63]. The avid- A nested RT-PCR assay, in which the RT-PCR product ity index (AI) is calculated according to the following formula from the first amplification reaction is re-amplified by internal [57]: primers, was developed and shown to provide a higher level of sensitivity for the detection of rubella virus RNA [72]. However, absorbance of avidity ELISA AI = 100 × the risk of contamination is markedly increased. The detection absorbance of standard ELISA limit of the RT-PCR assay is approximately two RNA copies. The AI is a useful measure only when the IgG concentration in Clinical specimens for rubella virus genome detection the patient’s serum is not below 25 IU [39]. Low avidity (usually include: products of conception (POC), CV,lens aspirate/biopsy, below 50%) is associated with recent primary rubella infection AF, fetal blood, pharyngeal swabs and spinal fluid (CSF) or while reinfection is typically associated with high avidity as brain biopsy when the central nervous system (CNS) is involved a result of the stimulation of memory B cells (immunological [68,69,73–75]. An additional advantage of RT-PCRis that it does memory) [39–41]. not require infectious virus [74]. RV is extremely thermo-labile In infants with CRS the low avidity IgG continues to be pro- and frequently is inactivated during sample transporation to the duced for much longer than in cases of postnatal primary rubella, laboratory. where it lasts 4–6 week after exposure [39]. This may be used Finally, it should be noted that clinical samples may con- for retrospective assessment of initially undiagnosed CRS cases. tain PCR inhibitors (such as heparin and hemoglobin), and the extraction procedure itself may cause enzyme inhibition 2.2.6. Rubella virus isolation in tissue culture [72,76,77]. This underscores the need and importance for strict Diagnosis of prenatal or postnatal rubella infections are internal quality control during each step of the RT-PCR proce- essentially based on the more reliable and rapid serological dure and participation in external quality assessment programs techniques. However, virus isolation is useful in confirming the is of a high value. diagnosis of CRS (Fig. 1) and rubella virus strain characterization required for epidemiological purposes. Rubella virus can 2.3. Summary be isolated using a variety of clinical specimens such as: respiratory secretions (nasopharyngeal swabs), urine, heparinized Rubella infection during pregnancy, although rare in coun- blood, CSF, cataract material, lens fluid, amniotic fluid, synovial tries with routine vaccination programs, is still a problem requir- fluid and products of conception (fetal tissues: placenta, liver, ing careful laboratory assessment. The laboratory testing should skin, etc.) obtained following spontaneous or therapeutic abor- confirm or rule-out recent rubella infection in pregnant women tion [6,36,44,64]. In order to avoid virus inactivation, specimens and identify congenital rubella infections in the fetus or neonate. should be inoculated into cell culture immediately or stored at Maternal infection is currently assessed by serological assays, 4 ◦C for not more than 2 days, or kept frozen (−70◦ C) for longer primarily by ELISA IgM and IgG. Borderline results for the IgG periods [36]. assay can be further assessed by the HI or NT assays available 360 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 in reference laboratories. Confirmation of recent infection can 3.1.2. Laboratory assessment of CMV infection in pregnant be sought using the IgG-avidity assay in addition to the other women tests. CMV was recognized as the cause of fetal stillbirth following Intra-uterine infection is assessed by IgM assays in fetal blood a cytomegalic inclusion disease (CID) in the mid 1950s when it which can be accompanied by virus detection in CV or AF spec- was first grown in tissue cultures in three laboratories [80–82]. imens, or, in case of induced abortion, in fetal tissue. Laboratory Since then demonstration of CMV infection of the mother or assessment of congenital rubella infection in neonates relies on fetus by laboratory testing has become an essential part of the virus detection by culture or RT-PCR in various clinical sam- assessment of pregnancies at risk [76,83]. Assessment of con- ples taken early after birth, and by demonstration of IgM and genital CMV infection begins with maternal serology which long-lasting IgG in neonatal serum. Due to the complexity of should establish recent primary or secondary infection (Fig. 2). the current laboratory assays, cooperation between the physi- Not all maternal infections result in fetal transmission and cian and the laboratory is of utmost importance to achieve a damage. Only 35–50% of maternal primary infections and reliable diagnosis. 0.2–2% of secondary infections lead to fetal infection, out of An algorithm describing the laboratory diagnosis process for which only 5–15% in primary infection and about 1% in sec- RV is shown in Fig. 1. ondary infections are clinically affected [84–87]. Therefore, following maternal diagnosis, and if early pregnancy termina- 3. Cytomegalovirus (CMV) tion was not chosen, subsequent prenatal diagnosis should take place using methods for virus detection in AF samples. 3.1. Introduction Demonstration of maternal infection relies on ELISA IgM and IgG assays and on CMV IgG avidity assay (Fig. 2). Unlike 3.1.1. The pathogen HI and NT for rubella, for CMV there are currently no serolog- CMV is a common pathogen which can cause primary and ical “gold standard” assays which can be used for confirmation secondary infections. CMV is a member of the herpesvirus fam- and reassurance. Recently an attempt to find association between ily possessing a 235 kb double stranded linear DNA genome, a viral load in maternal blood and the risk for fetal infection did capsid and a loose envelope. Membranal glycoproteins embed- not yield positive results [88]. ded in the envelope carry neutralization epitopes. CMV can infect all age groups usually causing mild and self-limited dis- 3.1.3. Prenatal assessment of congenital CMV infection ease. Its sero-prevalence in women of child-bearing age varies Maternal infection during pregnancy prompts testing for fetal from 50% to over 80%, with inverse correlation to socio- infection as outlined in Fig. 2. Prenatal CMV diagnosis can- economic levels. Primary CMV infection during pregnancy car- not rely on detection of fetal IgM since frequently the fetus ries a high risk of intrauterine transmission which may result does not develop IgM [76,89–94]. On the other hand, because in severe fetal damage, including growth retardation, jaundice, CMV is excreted in the urine of the infected fetus, detection of hepatosplenomegaly and CNS abnormalities. Those who are virus in the AF has proven to be a highly sensitive and reliable asymptomatic at birth may develop hearing defects or learn- method. Numerous studies have focused on the most appropriate ing disabilities later in life. It is now recognized that intrauterine timing for performing amniocentesis which will yield the best transmission may occur in the presence of maternal immunity sensitivity for detection of fetal infection [76,83,97–99]. These [78]. Pre-conceptional primary infection carries a high risk iden- studies clearly indicated that amniotic fluid should be collected tical to the risk of infection during early gestational weeks [79]. on 21–23 gestational week and at least 6–9 weeks past maternal CMV, like other members of the herpesvirus family, estab- infection. If these requirements are met then the sensitivity of lishes a latent infection with occasional reactivations as well as detection of intrauterine infection can reach over 95% while the recurrent infections in spite of the presence of immunity. How- general sensitivity is only 70–80%. One study measured the sen- ever, reactivation or recurrent infections carry a much lower risk sitivity for AF obtained at gestational weeks 14–20 and reported for fetal infection and damage is much lower in such events. only 45% [100]. Most of the studies state that the timing of the The infectious cycle in vitro takes 24–48 h while in vivo the amniocentesis is more critical for sensitivity than the laboratory incubation period for postnatal infection can last for 4–8 weeks. methods used to detect the virus in the AF. The incubation period for congenital infection is not known and Initially, virus isolation in tissue culture and its more sophisti- the gestational age of congenital infection is currently defined cated variation “Shell-Vial” technique (Table 1) were the leading by the maternal seroconversion, if known, which does not nec- laboratory methods for detection of CMV in amniotic fluid. essarily reflect the actual timing of the fetal infection. However, during the late 1980s highly sensitive molecular meth- The host defense against CMV infection in immune- ods were developed for detection of specific viral DNA in competent individuals combines cellular and humoral immune clinical specimens such as dot-blot hybridization [101–103]. responses which together prevent a severe CMV disease in the These methods were much faster, less laborious and repeat- vast majority of infections. Antibodies of the IgM class are able compared to virus culturing. Performance of the biological produced immediately after primary infection and may last for and molecular techniques in parallel assured that the precious several months. IgM can be produced in secondary infections amniotic fluid sample will not be wasted and that false negative in some cases. Antibodies of the IgG class are also produced results will not be obtained by a technical problem in any of immediately after infection and last for life. these “home-made” assays. E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 361

Since the early 1990s the polymerase chain reaction (PCR) since a “significant increase” is rarely defined for commercial has become the preferred method for CMV detection in amniotic ELISA assays, it is up to the laboratory to define it. fluid [95,96,104–106]. Problems with molecular contamination leading to false positive results and the need to address prog- 3.2.3. CMV IgG avidity assays nostic issues, led finally to the development of quantitative PCR The IgG avidity assay was developed to circumvent diagnos- assays with the highly advanced real-time PCR (rt-PCR) as the tic problems as described for rubella [128–130]. It is performed most updated method (Table 1). Current studies deal with the when both IgM and IgG are positive on initial testing, but cannot correlation between the “viral load” in the amniotic fluid and be performed on sera with very low IgG titers. Various commer- the pregnancy outcome, in an attempt to establish the prognos- cial assays are calibrated in different ways for determination of tic parameters of this powerful technique. the diagnostic threshold: some assays exclude recent infection The laboratory methods used for assessment of maternal and if the AI reaches a certain threshold level, yet others approve fetal CMV infection are described in detail below. recent infection if the AI is lower than a certain threshold level. However, none of these assays can exactly determine when the 3.2. Laboratory assays for assessment of CMV infection infection occurred or give any interpretation of results falling outside of its exclusion or inclusion criteria. Numerous studies 3.2.1. CMV IgM assays published in recent years aimed at evaluating IgG-avidity assays IgM detection is a hallmark of primary infection although it (by commercial kits or “in-house” methods) for their ability to may also be associated with secondary infections [90,107–109]. identify or exclude recent primary CMV infection, and to predict Major efforts were put into developing sensitive and reliable congenital infection. Concordance between different commer- assays for IgM detection using ELISA. The technical and bio- cial assays for determination of low avidity was high (98–100%), logical obstacles and their solutions which were described for but not for determination of high avidity (70%). Because the use rubella IgM assays apply for CMV as well, including long-term of this assay is relatively new, some of these studies are described persistence of IgM antibodies [110–114]. in detail below. The source of the viral antigen affects sensitivity and speci- One set of studies evaluated the ability of the assay to assess ficity [113,115–122], but in the absence of a gold standard assay, the risk for fetal infection. [127,131–133]. In a cohort of women comparisons between various commercially available assays considered at risk for transmitting CMV to their fetuses based were based on multi-variant analyses of “consensus” results on demonstration of IgM or seroconversion, low avidity was between several assays. These studies demonstrated high vari- strongly associated with fetal infection (100% sensitivity) if ability in specificity and sensitivity among assays and a high rate the serum sample tested was collected at 6–18 weeks gesta- of discordance [123–126]. Thus, testing for IgM, particularly in tion. Moderate or high AI levels were associated with 33% asymptomatic pregnant women, may frequently create a prob- and 11%, respectively, of cases with CMV genome-positive lem rather that solving it: borderline results or conflicting results amniotic fluid, but with no fetal infection. Lowered sensitivity among two or more commercial kits are interpreted as incon- (60–63%) for detection of primary infection was found for sera clusive and require further testing as described below. Other collected at 21–23 weeks gestation, since some of the mothers, methods, such as immunoblotting and IF assays (Table 1) were infected early in pregnancy, already developed moderate or high developed to confirm positive IgM results and to distinguish avidity. between specific and non-specific reactions [88]. However, these Another set of studies [134–136] examined the ability of the assays did not gain vast usage because of lowered sensitivity IgG-avidity assay to exclude those with past infection and there- [127] and the lack of automation. fore with low risk of fetal transmission. Women with positive or equivocal IgM but without documented seroconversion were 3.2.2. CMV IgG assays tested. High avidity was interpreted as remote infection which IgG assays which are currently based on ELISA, are generally did not occur within the last 3 months. The results of this series used for determination of immune status but, unlike rubella, of studies also showed that congenital infection was strongly there is neither definition of a CMV-IgG international unit (IU) associated with low avidity, while moderate or high avidity were nor of the protective antibody level. IgG assays may also help associated with uninfected fetus. Additional studies further con- to establish diagnosis of current CMV infection in suspected firmed the strong association between low avidity and primary secondary infections, or when the IgM result is inconclusive, infection, and thus risk for fetal infection, and between high by demonstration of IgG seroconversion or a significant IgG avidity and past infection [130,136–140]. One study showed rise between paired sera taken 2–3 weeks apart. This ability is the lack of full concordance between different commercial IgG limited in cases when women initially present with a high titer of avidity assays [141]. It showed that the ability of a commercial IgG or when it is impossible or too late to obtain a second serum kit to exclude recent infection by high avidity was restricted to sample. Commercial ELISA IgG assays are relatively simple, AI of >80% and to determine recent infection to AI of <20%. correlate well with each other and most of them are quantitative Any result in between those limits was inconclusive since sera but are not yet internationally standardized. Commercial kits use with AI of 50–80% included 48 out of 257 (18%) women with arbitrary units (AU) which differ from one assay to another and a history of past infection and 3 sera from 2 patients with a his- thus, to demonstrate an increase in antibody level, it is critical tory of recent infection. Testing the latter three samples with a to run the two samples in parallel in the same test. Additionally, different kit yielded low avidity (30%). 362 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382

In conclusion, the IgG-avidity assay is a powerful tool but except for rare cases in which the monoclonal antibody does not it should be used and interpreted properly. The association recognize the viral antigen [147–149]. between low AI and recent primary infection with a high risk Today, virus isolation from AF remains a key method for for congenital infection is stronger than the association between demonstration of fetal infection and has been described exten- moderate or high AI and past infection with low risk. Inter- sively in many studies either exclusively or in conjunction with pretable results can be achieved mainly for sera obtained within molecular methods, particularly PCR [97,99,132,150–155]. The the first 3–4 month of pregnancy. However both the inclusion main subject under investigation in recent years has been the and the exclusion approaches can be used and the IgG avidity comparative sensitivity and specificity of the PCR and the virus assay is now implemented in a testing algorithm following the isolation methods. IgG test [142] as shown in Fig. 2. 3.2.6. Detection of CMV by PCR 3.2.4. CMV neutralization assays Detection of viral DNA in clinical samples involves DNA Neutralizing antibodies appear only 13–15 weeks following extraction and analysis. PCR has become the preferred method primary infection, thus the presence of high-titer of neutralizing for rapid viral diagnosis in recent years. Its main disadvan- antibodies during acute infection indicates a secondary rather tage is the possible contamination leading to false positive than a primary infection. The neutralization assays have not results. reached a wide use as they are labor intensive, very slow and The PCR assay includes several components which can vary cannot be commercialized. Therefore, they are rarely performed from test to test. Viral DNA can be extracted using in-house by specialized reference laboratories. Attempts to correlate neu- methods or various commercial kits. The primers used can be tralization with specific response to the viral glycoprotein gB derived from different viral genomic sites and the reaction con- showed promising results and were also commercialized in an ditions can be altered. For CMV,most assays utilize the early (E) ELISA format [130,143–146]. However the utility of this assay or immediate-early (IE) genes which are highly conserved com- requires further studies. pared to the structural matrix or glycoprotein genes presenting higher variability among wild-type isolates. To increase sensitiv- 3.2.5. Virus isolation in tissue culture ity [95,156] some assays include a second round of amplification Virus is cultured from AF samples to assess fetal infection using nested or hemi-nested primers. The nested PCR is how- (Fig. 2). Since CMV is a very labile virus, samples for culturing ever more prone to molecular contamination and false-positive should be kept at 4–8 ◦C and transported to the laboratory within results. 48 h to be tested immediately. Freezing AF at −20 ◦C destroys The comparative specificity and sensitivity of PCR and virus virus infectivity and freezing at or below −70 ◦C requires stabi- isolation is dependent upon technical parameters which vary lization by 0.4 M sucrose phosphate [147]. from one laboratory to another with relation to the overall med- Culturing CMV from AF which was stored and transported ical set-up in which they are placed and the technical skills of appropriately, has always been considered the gold standard the laboratory personnel. However, it is generally agreed that for method for detection of fetal infection having 100% speci- CMV, PCR is more sensitive than tissue culture isolation. PCR ficity. CMV can be isolated in human diploid fibroblast cells is also a repeatable assay which is of great advantage in contro- either primary, such as human embryonic cells and human fore- versial cases. Original samples kept frozen at or below −70 ◦C skin cells, or continuous cultures such as MRC-5 and WI-38 can be re-processed and extracted DNA can be re-tested or sent cells [147]. The diploid cells should be used at low passage to another laboratory for confirmation. number to avoid loss of sensitivity. Tissue culture monolayers inoculated with the clinical samples should be maintained 3.2.7. Quantitative PCR-based assays for up to 6 weeks since CMV is a slow growing virus. Dur- Many previous studies have shown that detection of CMV ing this period blind passages should be performed using cells DNA in AF by itself does not predict the outcome of fetal infec- rather than culture supernatant since the virus is cell-bound. tion. Clinical measures such as ultrasonographic examinations CMV produces a typical CPE which can appear within 2–3 are a key component in fetal assessment, but might also fail to days and up to 6 weeks, depending on the virus concentra- detect affected fetuses. In an attempt to address prognostic issues tion in the clinical sample. The CPE is easily recognizable, it was suggested that symptomatic fetuses can be distinguished but IF assay using specific antibodies can confirm the presence from asymptomatic ones based on the viral load in the amniotic of CMV. fluid. Quantitative PCR methods were developed as “in-house” An alternative, much shortened procedure called the “shell- assays or are available as commercial kits using various tech- vial assay” was developed in which inoculated cultures are spun nologies. The most up-to date technology is the real-time PCR down at low velocity for 40–60 min before incubation at 37 ◦C. assay in which the amplified sequences are detected by a fluo- This procedure enhances and speeds-up viral infection of the rescent probe in a real-time and quantitative manner [157–159]. cultured cells. Infected cells are then detected within 16–72 h These assays, performed by dedicated instruments, carry the by IF using monoclonal antibodies directed against early viral advantages of high sensitivity and specificity conferred by the proteins synthesized shortly after infection. This method gained hybridization probe, and the lack of contamination by amplifi- wide acceptance and is now used by most laboratories. Its sen- cation products, since the reaction tubes are never opened after sitivity and specificity are highly comparable to virus isolation amplification. E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 363

Few recent publications have addressed the prognostic value and the fetus. Complications and sequelae include pneumo- of determination of viral load in AF with controversial results. nia, increased rate of prematurity abortions, congenital varicella Three studies [160–162] found no statistically significant differ- syndrome (CVS), neonatal varicella and herpes zoster during ence in viral load between symptomatic and asymptomatic fetal the first year of life [171–177]. Rarely VZV may cause a life infections. Yet other two studies reported predictive values for threatening CNS infection. The risk of adverse effects for the viral load [163,164]. In one of these two studies [164] the pres- mother is greatest in the third trimester of pregnancy, while for ence of 103 or more CMV genome-equivalents per millilitres the fetus the risk is greatest in the first and second trimesters. (GE/ml) predicted mother to child transmission with 100% prob- The risk of CVS for all pregnancies continuing for 20 weeks is ability, and 105 GE/ml or more predicted symptomatic infection. about 1%, but is lower (0.4%) between weeks 0 and 12 and is In the second report [163] CMV DNA load with median of higher (2%) between weeks 13 and 20. Maternal infection after 2.8 × 105 GE/ml was associated with major ultrasound abnor- 20 weeks and up to 36 weeks is not associated with adverse malities while median values of 8 × 103 GE/ml was associated fetal effect, but may present as shingles in the first few years with normal ultrasound and asymptomatic newborn. The slight of infant’s life indicating reactivation of the virus after a pri- discordance between the two studies calls for further evaluations mary infection. If maternal infection occurs 1–4 weeks before on a larger scale and underscores the need for standardization, delivery, up to 50% of the newborns are infected and 23% since the quantitative assays may vary by orders of magni- of them develop clinical varicella. Severe varicella occurs if tude using different methods or primers derived from different the infant is born within seven days of the onset of maternal genomic regions [165,166]. disease.

3.3. Summary 4.1.2. Assessment of VZV infection in pregnancy Laboratory diagnosis of VZV in pregnancy is required in two Laboratory testing for determination of intrauterine CMV situations: (a) the pregnant woman has developed clinical symp- infection involves several steps. Maternal primary or recurrent toms compatible with chickenpox or herpes zoster (Shingles) (b) infection is assessed by serology using IgM, IgG and IgG-avidity The pregnant woman was exposed to a chickenpox or a zoster assays. In controversial cases a second blood sample should be case (Fig. 3). sought to demonstrate antibody kinetics typical of current infec- If the pregnant woman has developed clinical symptoms tion and not of remote infection or a non-specific reaction. If the infection should be confirmed by laboratory testing using maternal primary infection was established and the pregnancy serology or virus detection by culturing, antigen detection or was not terminated, prenatal diagnosis follows at 21–23 weeks molecular methods. gestation and 6–9 weeks after seroconversion (if known). Detec- Assessment of VZV IgM which remains in the blood for 4–5 tion of CMV in AF is done by virus culturing and/or PCR. weeks is diagnostic. However, false positive results are com- Quantitative PCR is still not established for assessment of fetal mon in the presence of high VZV IgG antibodies and virus damage and prognosis. reactivations may also induce IgM. Therefore, determination During the diagnostic process, which may last for several of IgG seroconversion or a 4-fold rise in VZV IgG titer should weeks, collaboration between the laboratory and the physician accompany the IgM test. Virus isolation or PCR from dermal is of utmost importance. Appropriate timing of sampling, sam- lesions can be attempted and, if positive, confirm the diagnosis ple treatment, usage of validated assays under quality assessment (Fig. 3). conditions, and correct interpretation of the results are all essen- If the pregnant woman has reported exposure to a case of tial for obtaining a reliable diagnosis. chickenpox or zoster, prompt assessment of her immune status The algorithm describing the laboratory diagnostic process by testing for IgG within 96 h from exposure should be done for CMV is shown in Fig. 2. since varicella-zoster immunoglobulin (VZIG) given as a pro- phylactic measure at the time of exposure is known to prevent 4. Varicella-zoster virus (VZV) or reduce the severity of chickenpox [178,179]. Serological screening for IgG of women with negative or 4.1. Introduction uncertain histories of illness, who are planning a pregnancy, or of women who give history of recent contact with chicken- 4.1.1. The pathogen pox, has been suggested as a strategy for preventing CVS and Varicella-zoster virus (VZV) is a common pathogen belong- neonatal VZV [180,181]. In a study conducted in our labora- ing to the herpesvirus family which can establish latent infec- tory 52 pregnant women were assessed for immunity to VZV tions and subsequent reactivations. Primary infection, chicken- following exposure to chickenpox and 25% of them were found pox, is a common childhood disease. Reactivation is manifested susceptible. The attack rate among the susceptible women was as zoster and occurs in the presence of anti VZV antibodies. 85% [181]. In another study, Linder et al. [182] reported that Approximately 90% of the adult population is positive for VZV in 327 pregnant women assessed for VZV immunity, 95.8% of antibodies and studies on pregnant and parturient women found the women who recalled chickenpox in themselves and 100% of between 80% and 91% seropositivity [167–170]. women who recalled chickenpox in their children were seropos- Primary infection with VZV (chickenpox) during pregnancy itive, and only 6.8% of the women with a lack or uncertain carries a risk for clinical complications for both the mother history of exposure were seronegative. The screening strategy 364 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 might gain momentum due to the availability of VZV vaccine, 4.2.4. Virus isolation in tissue culture as seronegative women can be vaccinated. VZV isolation in tissue culture is not a very sensitive assay and the isolation of the virus from skin lesions is possible only 4.1.3. Prenatal and perinatal laboratory assessment of from early vesicles. VZV is a very labile virus which grows congenital VZV infection slowly in selected tissue cultures. Specimens for cell culture If VZV infection of the mother during the first or the sec- (vesicle fluid, skin-lesion swabs and AF) should be transported ond trimester has been confirmed, the need to diagnose the fetus to the laboratory as soon as possible after collection [196–198]. arises. Unfortunately, laboratory methods for fetal assessment Swabs should be put in a vial containing virus transport medium are of limited value. Assessment of fetal infection by determi- and AF should be placed in a sterile container. Both should be nation of VZV IgM in fetal blood is not widely performed. IgM kept at 4–8 ◦C during transportation. Inappropriate conditions may be manifested in the fetus only after 24 weeks of gesta- during transportation may easily reduce virus viability [199]. tion, thus in case of intrauterine infection during early gestation, Upon receipt, the specimen is inoculated into semi- functional immunity may not be present in the fetus [183]. continuous diploid cells such as human diploid fibroblasts Alternatively, determination of fetal infection can be done by (MRC-5) and continuous cell lines derived from tumors of demonstration of VZV DNA in AF using PCR, but its presence human or animal tissue such as A549. CPE may appear within 2 is not synonymous with development of CVS [183]. Only one weeks. AF should be sampled after 18 weeks of pregnancy and in 12 infected fetuses will develop pathological signs, so inter- after complete healing of skin lesions of the mother. Confirma- pretation of a positive VZV DNA result is problematic if the tion of virus isolation can be done by immunoflurescent staining fetus appears normal upon ultrasonograpic examination [180]. using monoclonal anti-VZV antibody. Shell vial cultures as Diagnosis of clinical varicella in neonates is based on serology described for CMV improve the sensitivity of VZV detection (IgM) and virus isolation or detection in vesicle fluid or in CSF and allow rapid identification of positive samples within 1–3 (in case of CNS infection). days [196,200].

4.2. Laboratory assays for assessment of VZV infection and 4.2.5. Direct detection of VZV antigen immunity Immunofluorescence or immunoperoxidase assays use monoclonal or polyclonal antibodies directed to VZV antigens to 4.2.1. VZV IgG assays detect VZV infection in epithelial cells isolated from AF, or Several methods were applied for determination of VZV from suspected lesions (Table 1). This simple method allows antibodies. In the past, specific IgG antibodies were measured rapid diagnosis (approximately 2 h) of VZV infection and is by the complement fixation (CF) assay, which can be used available to most laboratories [201,202]. It is highly specific and only for diagnosis of recent infection or by the latex agglu- has sensitivity ranging from 73.6% to 86%. The preferred mon- tination assay [184–188]. Today, highly sensitive and specific oclonal antibodies are those directed against the cell-membrane ELISA and immunofluorescence (IF) methods are used for associated viral antigens [203]. Stained smears, in which multi- determitation of VZV IgG antibodies. Several versions of the nucleated giant cells are seen are less sensitive, 60–75% [204]. IF assay exist (Table 1): fluorescent antibody to membrane antigen (FAMA) and indirect fluorescent antibody to membrane 4.2.6. Molecular methods for detection of viral DNA antigen (IFAMA) detect binding of antibodies to membrane PCR and hybridization methods to detect VZV DNA antigens in fixed VZV infected cells, and are highly specific sequences are very sensitive and specific [183,191,205–208]. and sensitive [169,181,186,189]. ELISA is comparable to IF Modifications of the basic PCR technique have been used to in sensitivity, specificity and cost and can be used for general increase the sensitivity by using nested PCR assays. However, screening purposes. Commercial ELISA kits for VZV IgG are this assay is highly susceptible to contamination, leading to false generally highly specific but demonstrate some false positive positive results. Rapid laboratory diagnosis is important when results compared to FAMA or IFAMA. the CNS is involved, especially in cases with clinically confused dermal manifestations, and is crucial in neonates to prevent lethal 4.2.2. VZV IgM assays outcome of disease. In recent years real-time PCR assays were Both the IF and ELISA methods are used for determination developed for VZV as for CMV and other viruses. Real-time of VZV IgM in the same manner as for IgG, except that the PCR assays appear to have equal sensitivity as VZV nested PCR conjugate is an anti-human IgM rather than anti-human IgG. assays but are faster, easier and have markedly reduced risk for Commercial ELISA IgM kits may give false positive results as molecular contamination. Because of its high sensitivity com- was described for RV and CMV. pared to other methods, PCR has become the most appropriate method for detection of VZV DNA in AF and other specimens 4.2.3. Virus detection in clinical specimens [183,206,209,210]. However, standardization is a major prob- Virological methods for the diagnosis of VZV infection lem and should be done in order to avoid false positive or false include detection of infectious VZV, viral antigens and viral negative results. DNA in clinical specimens. These include vesicular fluid, swabs In a study conducted in our laboratory AF samples were or smears, AF in pregnant women or cerebrospinal fluid (CSF) analyzed either by PCR or real-time PCR in parallel to tissue in encephalitis cases [183,190–195]. culture isolation. The DNA amplification target was located in E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 365 the viral UL gene. VZV DNA was detected in the amniotic fluid infections are associated with prematurity and fetal growth of two (7.4%) out of 27 women who developed chickenpox in retardation. the second trimester of pregnancy. One gave birth to a normal Herpes simplex is a devastating infection in the neonate, child while no follow-up was available for the second woman. with primary asymptomatic and symptomatic maternal infec- All amniotic fluid cell cultures were negative. No case of CVS tion near delivery carrying a greater risk to the newborn than occurred following negative PCR results (unpublished data). recurrent infections [218,219]. Fourty percent of women who acquired genital HSV during pregnancy but did not complete 4.3. Summary their seroconversion prior to the time of delivery will infect their newborns. Transmission of genital herpes during vaginal VZV infection during pregnancy poses a risk to the mother delivery is high in neonates exposed to asymptomatic shedding and fetus. Laboratory assessment of maternal infection is based [220–223], since serial HSV genital cultures during the last few on serology (both IgG and IgM), and on virus detection in weeks of gestation are no longer recommended as a method skin lesions. Exposure of a pregnant woman to a varicella case to prevent neonatal herpes. Instead, women are examined at the prompts immediate assessment of her immune status to deter- time of labour and caesarean section is carried out only if there is mine the need for VZIG administration. The availability of a an identifiable lesion. The use of fetal scalp electrodes may also VZV vaccine may encourage the screening of women for VZV increase the risk for neonatal infection [224,225]. HSV infec- immunity before conception. tions should be suspected as the cause of any vesicle appearing There are no fully reliable methods to assess fetal infection in the neonate. and damage. Fetal IgM and virus detection in AF are used, but false negative results are common and positive results do not 5.1.2. Laboratory assessment of HSV infection in necessarily correlate with fetal damage. pregnancy and in neonates An algorithm describing the laboratory diagnostic assays for Clinical symptoms compatible with genital herpes during VZV infection in pregnancy is shown in Fig. 3. pregnancy require laboratory assessment (Fig. 4). Diagnosis of HSV infection relies on both serological and virological meth- 5. Herpes simplex virus (HSV) ods, but the diagnostic process may be complicated due to the nature of the viral infection. Recurrent infections and reacti- 5.1. Introduction vations pose a special challenge to serology, and therefore viral detection in genital lesions is more reliable as a diagnostic mean. 5.1.1. The pathogen The standard laboratory method to confirm current HSV Herpes simplex virus (HSV) establishes latent infection fol- infection is virus isolation and typing in cell culture since HSV lowing primary infection which may lead to reactivation. It is grows readily in tissue culture. The virus may be isolated within a neurotropic member of the herpesvirus family and the genus 2–4 days from swabs taken from herpetic skin and laryngeal or consists of two types: HSV type 1 (HSV-1) and HSV type 2 genital lesions (Fig. 4). (HSV-2). PCR techniques improved the correct diagnosis of HSV The consequences of infection with HSV can vary from infections especially in cases without clear overt manifestations asymptomatic to the life-threatening diseases manifested as [226–229] and can detect viral DNA from lesions that are culture HSV-encephalitis and neonatal herpes [211]. Recurrent infec- negative. For genital swabs it might be too sensitive to reflect tions with one type after primary infection with the other type true reactivation, and it is not clear whether a positive PCR in are common. HSV-1 is usually transmitted through the oral route a patient with a negative HSV culture reflects a true risk of and causes disease in the upper part of the body, while HSV-2is a transmission of the virus. Therefore physicians should interpret sexually transmitted virus which tends to cause primarily genital results with caution and according to additional criteria. Supple- herpes. However, both HSV-1 and HSV-2 can cause genital her- mental serological testing can be used when genital lesions are pes which can be a severe disease in primary episodes [211–214]. not apparent. An increase in the prevalence of genital herpes infection has been In primary infections antibodies appear within 4–7 days after documented worldwide: HSV-2 was the main cause of genital infection and reach a peak at 2–4 weeks. Antibodies persist herpes during the 1980s but since the 1990s HSV-1 constitutes for life with minor fluctuations. Specific IgM antibodies appear an increasing proportion of the cases. This shift from HSV-2 to after primary infection but may be detectable during reccurent HSV-1 may have implications for prognosis [215]. Assessment infections as well [226]. HSV-1 and HSV-2 share cross reactive of risk and diagnosis of HSV infections may involve testing for epitopes of the surface glycoproteins which are the major targets both HSV-1 and HSV-2. of serum antibodies. Therefore it is difficult to identify a newly HSV infection has serious consequences for the fetus and acquired infection with one HSV type on the background of neonate. Before 20 weeks of gestation, transplacental trans- pre-existing immunity to the other type (usually infection with mission can cause spontaneous abortion in up to 25% of the HSV-1 precedes infection with HSV-2). Type specific serologi- cases [216,217]. Later in pregnancy HSV infections are not cal assays were developed [230] which may be valuable for the associated with increase in spontaneous abortions but intrauter- management of a pregnant woman and her partner: these assays ine infections may occur. Symptomatic and asymptomatic first can identify previous infections, seroconversions and discordant episodes of genital herpes but not asymptomatic recurrent couples. The results may provide information to the pregnant 366 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 woman about her risk of acquiring and transmitting HSV to her for HSV is the most accurate method for type-specific serology infant (Fig. 4). The cost-benefit value of general screening for [241]. This method is the serology gold standard and is per- type-specific antibodies in pregnant women and their partners formed only by few laboratories. WB is expensive to perform were recently assessed [231,232]. and requires 2–5 days for completion. New approaches to anti- Rapid and sensitive diagnosis of neonatal HSV disease gen preparation include the application of recombinant gG-1 and is of utmost importance for initiation of acyclovir treatment gG-2 from a mammalian expression systems to nitrocellulose, and improvement in the outcome of neonatal herpes has been or purification of glycoprotein gG2 by lectin chromatography achieved due to the application of PCR as a diagnostic tool [233]. [242]. Neonatal HSV infections are confirmed through positive viral culture or PCR from skin, eye lesion, nasopharynx, rectum or 5.2.3. HSV IgM assays CSF and detection of IgM in a neonate is highly significant These assays are based primarily on ELISA and IF methods [234]. Neonatal HSV infection can occur in newborns without as described for VZV. Due to the persistent nature of the viral the presence of any vesicular skin disease [229,235,236]. infection and the frequency of recurrent infections, the presence of IgM is of high value primarily in neonates and infants. In most 5.2. Laboratory assays for assessment of HSV infection and cases testing for IgM must be accompanied by testing for IgG, immune status since a negative IgM result does not rule out recent infection.

5.2.1. HSV IgG assays 5.2.4. Virus isolation in tissue culture Several techniques are used for detection of HSV specific HSV is a labile virus, and successful virus culturing from IgG, among them CF, NT, IF and ELISA. The principles of clinical specimens depends on appropriate sample collection and these assays were described above for VZV and are shown in maintenance of the cold chain. Swabs should be placed into viral Table 1. There are assays which do not distinguish between IgG transport medium and other samples should be placed in a sterile directed to HSV-1 or HSV-2. These assays are useful in primary container. Rapid transportation of specimens to the laboratory infection in the absence of prior immunity, and they use crude and avoiding freeze-thawing cycles results in more than 90% preparations of cells infected with HSV-1 and/or HSV-2. They sensitivity of culturing from skin lesions. However, poor sample may detect infection with HSV-2 on the background of HSV- quality due to inappropriate sampling or compromised transport 1 [237]. More accurate type-specific assays were developed in conditions may reduce sensitivity by 20% for primary episodes order to detect recurrent infections with either type. and down to 50% for recurrent episodes even if herpetic lesions are present. When an appropriate sensitive cell line (such as 5.2.2. HSV type-specific IgG assays Vero cells) is used, appearance of a characteristic CPE within Type specific serological assays vary in their abilities [230]. 18–96 h after inoculation points to the presence of active HSV NT antibody level can be measured specifically for each type by [237]. Direct immuonofluorescence detection of HSV in cultures the plaque reduction assay using serial dilutions against a fixed with CPE is regarded as the standard for confirmation of HSV dose of HSV-1 or HSV-2, with 50% reduction in the plaque presence. number as an endpoint. However these assays are accurate only The so-called “Shell vial” method, described earlier for CMV in patients infected with only one virus type. NT assays were isolation (chapter 2.5 and Table 1) shortens the time required used for determination of HSV-2 infection based on the ratio to identify HSV in specimens to 24–48 h. An innovated com- between the antibody titers to the two viruses. Positive cases mercially available diagnostic test for rapid detection of HSV in were those with HSV-2 to HSV-1 ratio of ≥1 [237,238]. tissue culture is the ELVIS HSV test kit. It utilizes a recombinant IF can be used for detection of antibodies to HSV cell sur- cell system in which a reporter enzyme is quickly accumulated face and internal proteins. IF correlates well with NT titers but inside the infected cells and is detected by intense blue color. The subtyping is difficult. ELISA assays utilize a variety of condi- test is sensitive and specific and detects both HSV-1 and HSV-2 tions and antigens. These tests are more sensitive than NT and [243]. The kit is expensive and is used only by few laboratories. IF and can detect antibodies within the first week of infection. Manufacturers of those tests use mathematical calculations to 5.2.5. Direct antigen detection of HSV infer the presence of HSV-1 or HSV-2 antibodies based on their Other rapid assays such as the direct fluorescent assay (DFA) relative ratios. New type-specific tests, which distinguish HSV- or enzyme immunoassays (EIA) performed directly on smeared 2 from HSV-1 are based on glycoproteins gG1 and gG2 [239] cells recovered from lesions are rapid but less sensitive than and can provide useful information on the etiology of the genital virus culturing. Accordingly, for detection of virus in genital herpes in the symptomatic patient when viral culture and PCR swabs they may have similar sensitivity to viral culture for are not helpful. These tests allow the identification of patients symptomatic shedding but have reduced sensitivity for detecting with unrecognized genital herpes [240,241]. There are few good asymptomatic viral shedding. commercial ELISA kits for type specific serology which were approved by the FDA. Nevertheless, HSV type specific antibody 5.2.6. Detection of HSV DNA by PCR tests are interpreted in the context of the clinical history, clin- Methods for detection of HSV DNA, particularly PCR tech- ical presentation and other laboratory test results for herpes or niques (PCR, nested PCR and real-time PCR) were described other possible etiological agents. Finally, Western blotting (WB) above for other viruses. They are highly sensitive especially for E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 367 samples with very low concentration of virus, as was demon- [261,262] and fetal infection may lead to severe anemia, general- strated by numerous research studies. The performance of PCR ized edema, congestive heart failure, myocarditis, fetal hydrops analysis is dependent upon the quality of the specimen. The and fetal death [263–267]. Fifty percent of women at childbear- reported sensitivity for PCR is 75–100% for the diagnosis of ing age are susceptible to parvovirus B19 infection as judged CNS HSV infection [228,244,245]. The reason for the broad by the lack of IgG antibodies to the virus [267–272].How- range in sensitivity is the differences in methodologies and ever, transplacental transmission rates have been estimated at variability in performance of PCR between laboratories. Stan- between 25% and 33%, and the incidence of fetal loss is con- dardization using reference samples to assure identical results sidered between 1.66% and 9% [271,273]. Since intrauterine is not yet in place [227]. blood transfusions can increase fetal survival rate if done early enough, it is critical to diagnose maternal and fetal infection 5.3. Summary accurately and timely, and laboratory testing is an essential part of the diagnosis [274–279]. HSV-2 is the main type involved in congenital and neonatal infection, although genital HSV-1 infections have become more 6.1.2. Laboratory assessment of parvovirus B19 infection common in recent years. Primary maternal symptomatic and in pregnancy asymptomatic infections may cause a serious risk of abortion Laboratory diagnosis of parvovirus B19 infection has been and fetal growth retardation, while infection during delivery may complicated by the nature of the virus, the viral infection pro- cause a devastating neonatal disease. cess and the immune response. Parvovirus B19 is a fastidious Laboratory assessment of maternal infection and immune sta- virus which cannot be grown in regular continuous cell lines. In tus as well as neonatal infection is critical for timely management those cells where it grows it replicates poorly and the virus yield and treatment. Virus isolation from genital lesions is the primary is minimal, thus viral culturing is not a diagnostic option and tool for assessment of maternal infection. Serological asays are cannot be used for NT assays or for preparation of native viral useful but are complicated by pre-existing heterotypic antibod- antigens for serology [261,280,281]. ies. Type-specific IgG assays must be applied for determination Assessment of parvovirus B19 infection in pregnant women of recurrent maternal infections. relies primarily on serology (Fig. 5). During acute infection IgM Virus isolation, antigen detection or DNA detection by PCR antibodies appear 7–14 days from infection with Parvovirus B19 in skin lesions samples are additional tools for rapid and sensitive and may last for 6 month or even longer. IgG antibodies appear diagnosis of symptomatic current infection. several days after IgM and persist for life. The antibodies pro- Prenatal diagnosis is not common and has not been assessed duced specifically react with both structural and non-structural for sensitivity and specificity. Neonatal infection is diagnosed viral proteins and recognize linear and conformational epitopes by virus culturing or PCR in skin lesions or other clinical speci- of the capsid proteins VP1 and VP2 [282–288]. The antibodies mens in case of a disseminated disease, and by detection of IgM recognizing conformational epitopes last longer for both IgM antibodies. [285] and IgG [282,284,286], and IgG antibodies recognizing An algorithm describing the laboratory diagnostic assays for linear epitopes disappear around 6 month post infection. This HSV infection in pregnant women and neonates is shown in creates technical problems for serological assays as described Fig. 4. below. Maternal infection during pregnancy is frequently asymp- 6. Parvovirus B19 tomatic and by the time of fetal infection and symptoms, 2–12 weeks after maternal infection, she may have high IgG and 6.1. Introduction no IgM responses [260,275,289,290]. Viral DNA detection by highly sensitive molecular assays such as dot-blot hybridiza- 6.1.1. The pathogen tion and PCR can be applied to maternal serum or whole blood Human parvovirus B19 (B19) can infect humans at all ages. [276,291]. However, parvoviruses are known for their ability to It is a member of the family Parvoviridae, genus Erythrovirus integrate into the host genome and to establish persistent infec- which comprise small, non-enveloped viruses containing linear tions [292–296]. Recent studies [297–299] showed that B19 single-stranded DNA genomes. Most of the humoral immune DNA can be detected in serum samples by highly sensitive meth- response is directed to the two capsid proteins VP1 and VP2 ods up to 6 months after acute phase viremia, although quanti- [246]. The seroprevalence is 50% by age 15 and exceeds 80% by tative assays showed that the amount of DNA present decreased age 70 [247]. Childhood infection is mild leading to the disease with time [300]. It has also been shown that 0.01–0.03% of the erythema infectiosum (“fifth disease”) [248–251] In adults the healthy blood donors have B19 DNA detected in their serum clinical symptoms vary from asymptomatic or mild to severe [301–303]. Thus, detection of B19 DNA in maternal blood by illness including exanthematous disease, arthropathies, aplastic PCR might not be related to recent infection. crisis and, in immunocompromised patients, to chronic anemia [252–260]. 6.1.3. Prenatal laboratory assessment of congenital B19 Maternal infection during pregnancy may lead to fetal infec- infection tion since viremia is very high and lasts for 6–8 days. The In many cases congenital B19 infection is suspected only virus replicates in erythroid precursor cells and fetal tissues when typical fetal abnormalities are observed. Since the fetus 368 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 does not always develop IgM it is necessary to detect the virus serum samples from blood donors with no IgM were tested. itself in fetal samples. B19 cannot be cultivated and therefore Overall, 353 sera were found positive by all methods combined. detection of viral DNA is sought. The type of specimen and Of those 98.6%, 94.6% and 89% were positive by assays a, b the detection methods are still controversial: fetal blood and AF and c, respectively. Some sera reacted only with conformational are the most common specimens obtained and the most highly epitops. The results of this study underscore the need to use sensitive molecular methods are employed. However, 10–25% ELISA-IgG assays which include conformational epitopes of of the specimens from asymptomatic fetuses may have B19 DNA both VP1 and VP2 or VP2 alone, and demonstrate the lower [289,290]. This drives the need to develop quantitative molecular sensitivity of the Western blot assay. assays which can determine viral load and possibly associate In conclusion, when evaluating recent or past infections in it with the risk to the fetus. Those assays include quantitative pregnant women it is important to use well-established ELISA PCR-ELISA, in situ hybridization and rt-PCR. The latter has assays, and in cases of discrepancy between the test results and advantages which will be discussed below [304–308]. the clinical and epidemiological circumstances, confirmation must be sought using other commercial assays as well as sup- 6.2. Laboratory assays for assessment of parvovirus B19 portive molecular assays discussed next. infection 6.2.2. Detection of viral DNA in maternal and fetal 6.2.1. B19 IgM and IgG assays specimens Several commercial tests have been developed on the basis of Due to the limitations of the serological assays described recombinant and synthetic antigens [309–311]. These recombi- above and since viremia is long-lasting in B19 infections, molec- nant antigens frequently lack important conformational epitops ular assays for detection of B19 viral DNA were developed early. reducing the sensitivity and specificity of the assays. More- The methods most frequently used are DNA hybridization and over, “gold standard” methods like virus NT or HI assays are PCR. absent, and it is difficult to assess the sensitivity and specificity Molecular hybridization assays employing DNA probes of the serological assays. The variation in specificity and sensi- derived from most of the viral genome are labeled with 32P, tivity among commercial assays is high, and discordant results biotin or digoxigenin [319–322] and are capable of detecting are often obtained indicating false positive and false negative approximately 104–105 genome copies/ml [275,323]. This assay responses [314–317]. is sensitive enough to detect viral DNA in serum during peak Studies aimed at evaluating the sensitivity and specificity of viremia since the viral load exceeds 1010 genome copies/ml, various such assays were mostly based on comparative evalu- but low levels of viremia may be missed [275,296,298]. The ation, using samples selected on the basis of clinical diagno- PCR assays, developed later relied on a variety of primer sis. In a study conducted in Norway [315], five commercial sets derived from different genomic regions and were capa- ELISA and IF IgM kits were comparatively evaluated in three ble of detecting 102–105 genome copies/ml [324,325]. The groups of patients. The calculated specificities for the kits were nested PCR, the PCR-ELISA and the PCR-hybridization assays 70.1–94.8%, but the authors refrained from determining sensi- increased sensitivity and moved the detection limit down to tivity because of the absence of suitable reference methods. In 1–10 genome copies/ml [291,292,296,297,299,326–328].By another study conducted in Sweden [316], four commercial IgM using those highly sensitive methods it was revealed that in assays were evaluated in comparison to an IgM antibody capture many cases serology failed to detect maternal or fetal infec- radioimmunoassay as a reference method [318]. The calculated tions, as some seronegative mothers as well as fetuses turned sensitivity varied between 90% and 97% and the specificities out positive in the DNA detection assays. This finding was not varied from 88% to 96%. surprising in view of the known limitations of the serological In a third study conducted in the USA [314] three ELISA sys- assays. The ability to detect maternal and fetal infection by the tems utilizing one or more conformational antigens for detection highly sensitive molecular methods allowed correct diagnosis of of B19 IgM or IgG in sera of 198 pregnant women were compar- previousely unresolved cases, particularly cases of intrauterine atively evaluated. Agreement with the consensus results varied infection which did not result in fetal hydrops or were completely from 92.3% to 100% for IgM and from 97.9% to 99.5% for IgG. subclinical [275,276,290,292,296,297,324,327,329]. However, The authors note the high agreement in this study compared to as stated above, low levels of B19 DNA in maternal blood can their previous study [312] relating to the fact that the antigens be unrelated to recent infection. utilized in the two studies were substantially different: the ear- The use of various techniques without standardization, the lier study used a linear VP1 antigen while the latter one used a epidemiologically variable circumstances (epidemic VS non- conformational VP2 antigen. epidemic years), and the use of different primer sets and clinical A study designed to evaluate the most appropriate assays for specimens led to a high variability in the sensitivity, specificity IgG detection was conducted in Italy [313] which compared the and interpretation of the assays results. Moreover, new geno- performance of three commercial assays using different anti- types discovered recently were missed by common primer sets. gens: (a) an ELISA assay using VP1 + VP2 recombinant native Comparison of IgM and DNA detection by PCR in fetal blood conformational antigens, (b) an ELISA assay using VP2 recom- was conducted in a study done on 57 pregnant women and their binant native conformational epitopes, (c) a Western blot assay fetuses who had abnormal ultrasonography [327]. Viral DNA using denatured linear antigens. Four hundred and fourty six was found by PCR in 16 out of 58 fetuses (27%) while IgM was E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 369 detected only in 7 (12.3%). Two fetuses had false-positive IgM 6.3. Summary result, not supported by any other findings. Other researchers [276] compared maternal IgM to fetal serum or AF PCR results Parvovirus B19 infection during pregnancy may cause fetal in 56 women at high risk for B19 infection. They found pos- damage resulting in fetal loss. Early diagnosis of maternal infec- itive PCR in 24 IgM-negative/IgG-positive and 4 seronegative tion will allow fetal assessment and treatment by intrauterine (total 50%) out of, in addition to positive PCR in 15 (26%) of blood transfusion. Unfortunately, mothers often are not aware IgM-positive women. Another group [282] reported detection of of their infection until fetal damage is observed. viral DNA in fetal serum or AF by a PCR-hybridization assay Confirmation of B19 infection requires laboratory assess- in 11 out of 80 cases of fetal hydrops (14%) while maternal IgM ment, which is complicated by the nature of the viral infection antibodies were detected only in 3 (3.7%). Finally, a compre- and immune response. Serology, performed by using ELISA hensice prospective study in 18 fetal hydrops cases conducted assays rely on recombinant antigens and concordance is low in Italy [275] examined maternal serum, fetal cord blood and among all commercial assays available. In the absence of a “gold amniotic fluid using nested PCR, dot-blot hybridization and in standard” assay false positive and false negative results prevail. situ hybridization (ISH). The results showed that the ISH assay Virus culturing is impossible and virus detection is based on in fetal blood cells was 100% sensitive while the other methods various molecular assays. missed few to many cases. The conclusion drawn from this study In spite of several studies there is no consensus regarding the is that various assays have complementary roles, and reliable most appropriate clinical specimen and method for detection of diagnosis can be achieved only by a combination of serologi- viral DNA. Currently, on practical grounds, it is recommended cal and molecular assays done on maternal and fetal samples as to use ELISA IgM and IgG assays based on recombinant con- outlined in Fig. 5. formational epitopes of VP1 and VP2 or VP2 alone, and to use AF or fetal serum for detection of fetal infection by the most 6.2.3. Quantitative assays for detection of viral DNA sensitive molecular methds available (nested PCR or rt-PCR). The need to determine the quantity of B19 viral DNA present Since B19 may establish long lasting infection in the absence in clinical samples (viral load) arose because B19 can establish of symptoms, interpretation of viral DNA detection in mater- long-lasting persistent infection in immunocompetent individ- nal blood is difficult. Assessment of fetal infection and risk uals. It is not clear if low viral loads reflect whole genomes should rely on the clinical situation and other prenatal diagnostic or viable infectious virus particles, or only pieces of viral DNA. means. Studies in seronegative plasma-pool recipients showed that only An algorithm describing the most practical approach to lab- recipients of plasma containing >107 genome copies/ml became oratory assessment of B19 infection in pregnancy is shown in infected or seroconverted [305]. B19 DNA can also be detected Fig. 5. in solid organ tissues by PCR for years [323,330,331]. As noted earlier viral DNA can be detected in fetal tissue or blood in 7. Human immunodeficiency virus the absence of any detectable congenital abnormalities, and the outcome of detectable fetal infection, including fetal hydrops, 7.1. Introduction varies from spontaneous resolution to still-birth. Real-time PCR assays developed recently are equivalent to 7.1.1. The pathogen or more sensitive than nested PCR and PCR-ELISA but are Human immunodeficiency virus (HIV) is a retrovirus that much less prone to molecular contamination and produce a infects helper T cells of the immune system causing a progressive quantitative result which can be standardized and automated reduction in their numbers, and eventually acquired immunode- [304,306–308,332]. In a retrospective study, Knoll et al. [332] ficiency syndrome (AIDS). HIV is a member of the Retroviridae used real-time PCR to investigate the viral load in paired samples family [333], genus Lentivirus (or “slow” viruses). The course from mothers and their abnormal fetuses, and from mothers with of infection with these viruses is characterized by a long interval normal fetuses who were exposed to B19. The viral load in the between initial infection and the onset of serious symptoms. The maternal serum ranged from 7.2 × 102 to 2.6 × 103. The authors single-stranded RNA viruses exploit their reverse transcriptase did not report statistically significant correlation between mater- enzyme to synthesize DNA using their RNA as a template. The nal or fetal viral load and fetal condition. However, they noted DNA is then incorporated into the genome of infected cells. that they could not exclude a correlation between peak mater- AIDS was first diagnosed in European sailors with African nal viremia levels and fetal condition since most of the mothers connections [334–336]. The first AIDS virus, HIV-1, was ini- were not aware of their infection until onset of fetal symptoms, tially identified in 1983 [337–341]. A second AIDS virus, HIV-2, and their serum was collected after peak viremia. Although the was discovered in 1986 [342–344]. Forty million people were viral load in fetal sera was higher than in AF samples, the rt- estimated to be infected with HIV at the end of 2004 [345]. The PCR assay detected all positive cases using either one of these highest prevalence is found in Sub-Saharan Africa and it is rising fetal specimens. The authors recommend testing AF rather than mainly in Asia and some of the former Soviet Union countries fetal blood because AF can be drawn earlier, is simpler to obtain like Ukraine and the Russian Federation [345,346]. During its and less risky to the fetus. Clearly, more prospective studies are spread among humans, group M HIV-1 (one of three groups: M, necessary on larger groups of patients to learn more about the O and M), has evolved into multiple subtypes that differ from association between viral load and pregnancy outcome. one another by 10–30% along their genomes [347–350]. 370 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382

HIV is passed on primarily via four routes: unprotected Knowledge of maternal HIV infection during the antena- sexual intercourse (both homosexual and heterosexual), shar- tal period enables HIV-infected women to receive appropriate ing of needles by IV drug users, medical procedures using antiretroviral therapy during pregnancy, during labor, and to HIV-contaminated blood, tissues or equipment, and mother- newborns to reduce the risk for HIV transmission from mother to-child transmission (MTCT). The likelihood of transmission to child [370,383,384]. It also allows counseling of infected is increased by factors that may damage mucosal linings of women about the risks for HIV transmission through breast exposed tissues, especially by other sexually transmitted dis- milk and advising against breast feeding in countries where safe eases that cause ulcers or inflammation. alternatives to breast milk are available [385]. Early diagnos- HIV can be transmitted from infected mothers to infants dur- tic evaluation of HIV-exposed infants permits early initiation ing pregnancy (intrauterine) through transplacental passage of of aggressive antiretroviral therapy in infected infants and ini- the virus [351], during labor and delivery (intrapartum) through tiation of prophylaxis against Pneumocystis carinii pneumonia exposure to infected maternal fluids (blood or vaginal secretions) (PCP) in all HIV-exposed infants beginning at age 4–6 weeks in [352–355] and during the post partum period through breast- accordance with Public Health Services (PHS) guidelines [386]. feeding [356–359]. Infants who have a positive virologic test (see below) at or before age 48 h are considered to have early 7.1.3. Prenatal laboratory assessment of HIV infection (i.e., intrauterine) infection, whereas infants who have a negative Prenatal laboratory assessment of congenital HIV infection in virologic test during the first week of life and subsequent posi- AF or cord blood is not recommended as the invasive procedures tive tests are considered to have late (i.e., intrapartum) infection increases the risk of transmitting the virus from the maternal to [360]. In the absence of breast-feeding, intrauterine transmission the fetal blood stream. accounts for 10–35% of infection, and 60–75% of transmission occurs during labor and delivery [361–363]. Without interven- 7.2. Laboratory assessment of HIV infection tion, maternal infection leads to about 25–30% of babies being infected [364–369]. 7.2.1. HIV antibody assays Use of anti-retroviral therapy (ART) during pregnancy in The initial screening for HIV infection in adults and chil- HIV-infected women is associated with improved obstetric out- dren is done by testing for antibodies. Viral load assays are come of reduced infection rates of babies to less than 2% not intended for routine diagnosis but could be used in clini- [370–373] and little maternal toxicity [374–376]. The current cal management of HIV-infected persons in conjunction with US guidelines are to offer all pregnant HIV-1-infected women clinical signs and symptoms and other laboratory markers of highly active antiretroviral therapy (HAART) to maximally sup- disease progression. Detection of HIV-1 p24 antigen (Table 1) press viral replication, reduce the risk of prenatal transmission, is used for routine screening in blood and plasma centers but and minimize the risk of development of resistant virus. In their routine use for diagnosing HIV infection in individuals addition, HIV-infected women are offered an elective caesarean has been discouraged because the estimated average time from section delivery [355]. The results also suggest that special atten- detection of p24 antigen to detection of HIV antibody by stan- tion should be given to women belonging to previously identified dard enzyme-immuno-assay (EIA) is 6 days, and not all recently risk groups. infected persons have detectable levels of p24 antigen [387].In Because testing has proven very successful in helping to pre- the USA several FDA approved tests are available that enable vent the spread of the disease to babies, a US federal panel has the testing of HIV antibodies in different body fluids such as recommended that all pregnant women, not just those consid- whole blood, serum, plasma, oral fluid and urine. ered at high risk, be screened for the AIDS virus [377–381].No The standard testing algorithm for HIV-1 consists of initial prenatal (intrauterine) diagnosis is performed because: (a) the screening with an EIA to detect antibodies to the virus. Reactive infection can occur during delivery and (b) the testing interven- specimens undergo confirmatory testing with a more specific tion may increase the chance of virus transmission to the baby. supplemental test, usually a Western blot assay (WB) or, less Assessing the infection status of the mother is critical [381] and commonly, IFA (Table 1) [388]. Using both tests increases accu- following delivery the new-born should be tested. The laboratory racy of the results while maintaining their sensitivity [389–391]. testing is essential for the diagnosis. Only specimens that are repeatedly reactive by EIA and positive by IFA or reactive by WB are considered HIV-positive and 7.1.2. Importance of laboratory assessment of HIV indicative of HIV infection [389,390,392,393]. infection in pregnancy Incomplete antibody responses that produce negative or inde- Identification of HIV-infected women before or during preg- terminate results on WB tests can occur among persons recently nancy is critical to providing optimal therapy for both infected infected with HIV who have low levels of detectable antibodies women and their children and to preventing perinatal trans- (i.e., seroconverting), persons who have end-stage HIV dis- mission (see below). For women with unknown HIV status ease, and perinatally exposed but uninfected infants who are during active labor, ART can still be effective when given during seroreverting (i.e., losing maternal antibody). Non-specific reac- labor and delivery, followed by treatment of the newborn [382]. tions producing indeterminate results in uninfected persons have This expedited intervention requires the use of rapid diagnos- occurred more frequently among pregnant women than among tic testing during labor or rapid return of results from standard other persons [389–391,394]. testing. False-positive WB results are rare [395]. E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382 371

7.2.2. Detection of viral DNA in maternal and newborn antigen testing alone is not recommended because of its sub- specimens stantiallty reduced sensitivity and specificity compromising the 7.2.2.1. Diagnosis of HIV infection in maternal specimens. critical need for timely diagnosis [405]. Pregnant women are screened for the presence of antibodies Whether the current, more intensive antiretroviral combina- using the standard testing algorithm for adults (EIA plus WB or tion regimens women may receive during pregnancy for treat- IFA) as shown in Fig. 6. For women with unknown HIV status ment of their own HIV infection will affect diagnostic test sen- during active labor, rapid diagnostic tests are used if rapid return sitivity in their infants is unknown. Similarly, if more complex of results from standard testing is not available. regimens are administered to HIV-exposed infants for perinatal prophylaxis, the sensitivity of diagnostic assays will need to be 7.2.2.2. Diagnosis of HIV infection in newborns. Infant HIV re-examined [401]. testing should be done as soon after birth as possible so appropriate treatment interventions can be implemented quickly 7.2.2.3. Different subtypes. HIV subtype B is the predominant [377,384]. The standard antibody assays used for older children viral subtype found in the U.S. and western Europe. Non-subtype and adults are not useful for diagnosing children younger than B viruses predominate in other parts of the world, such as sub- 18 months as the presence of maternal antibodies makes sero- type C in regions of Africa and India and subtype E in much of logic tests uninformative. Therefore, a definitive diagnosis of southeast Asia. HIV infection in early infancy requires viral diagnostic assays, Currently the available HIV DNA PCR commercial tests are including HIV-1 p24 antigen assays, nucleic acid amplification less sensitive for detection for non-subtype B HIV, and false (e.g., PCR) or viral culture. HIV infection can be definitively negative HIV DNA PCR assays have been reported in infants diagnosed in most infected infants by age 1 month and in vir- infected with non-subtype B HIV [406–410]. Caution should be tually all infected infants by age 6 months. HIV infection is exercised in the interpretation of negative HIV DNA PCR test diagnosed by at least two positive assays using two separate results in infants born to mothers who may have acquired an HIV specimens [378]. non-B subtype. Some of the currently available HIV RNA assays HIV DNA PCR is the preferred virologic method for diag- have improved sensitivity for detection of non-subtype B HIV nosing HIV infection during infancy. It has 99% specificity to infection [411–413], although even these assays may not detect identify HIV proviral DNA in peripheral blood mononuclear some non-B subtypes, particularly group O HIV strains [414].In cells (PBMC) obtained from whole blood samples collected in cases of infants where non-subtype B perinatal exposure may be EDTA-containing tubes [396]. suspected and HIV DNA PCR is negative, repeat testing using A meta-analysis of published data from 271 infected children one of the newer RNA assays shown to be more sensitive for indicated that HIV DNA PCR was sensitive for the diagnosis of non-subtype B HIV is recommended (for example, the Amplicor HIV infection during the neonatal period. Thirty-eight percent HIV-1 monitor test 1.5, Nuclisens HIV-1 qt or Quantiplex HIV of infected children had positive HIV DNA PCR tests by age RNA 3.0 (bDNA) assays). In children with negative HIV DNA 48 h, 93% by age 14 days and 96% by age 28 days. No sub- PCR and RNA assays but in whom non-subtype B infection stantial change in sensitivity during the first week of life was continues to be suspected, the clinician should consult with an observed, but sensitivity increased rapidly during the second expert in pediatric HIV infection and the child should undergo week. close clinical monitoring and definitive HIV serologic testing at Quantitative assays that detect HIV RNA in plasma (see Sec- 18 months of age. tion 7.2.2.3 below) appear to be as sensitive as HIV DNA PCR for early diagnosis of HIV infection in HIV-exposed infants 7.2.2.4. Test algorithm for neonates. HIV infection is diag- [397–402]. The specificity is comparable between the two tests, nosed by two virological tests performed on separate blood but results of HIV RNA load below 104 copies/ml should be samples, regardless of age. The testing rules are summarized interpreted with caution [403]. Some clinicians use HIV RNA below and shown in Fig. 6: assay as the confirmatory test for infants testing HIV DNA PCR positive since it provides viral load measurement which guides (1) Initial testing is recommended by age 48 h. As many as treatment decisions. Available quantitative RNA tests include 40% of infected infants can be identified at this time. Blood the Amplicor HIV-1 monitor test 1.5 (Roche Diagnostics), the samples from the umbilical cord should not be used for diag- NASBA EasyQ HIV-1 (BioMerieux), the Quantiplex HIV RNA nostic evaluations, because of concerns regarding potential 3.0 (bDNA) (Bayer) and the LCx HIV RNA quantitative assay contamination with maternal blood. (Abbott Laboratories) assays [416–421]. However, special atten- (2) Repeated diagnostic testing can also be considered at age tion should be taken where non-B HIV is expected. 14 days in infants with negative tests at birth. The diag- HIV culture for the diagnosis of infection has a sensitivity nostic sensitivity of virological assays increases rapidly by that is similar to that of HIV DNA PCR [404]. However, HIV age 2 weeks. Early identification of infection would permit culture is more complex and expensive to perform than DNA discontinuation of neonatal ZDV chemoprophylaxis and a PCR, and definitive results may not be available for 2–4 weeks. further evaluation of the need for more aggressive drug com- Both standard and immune-complex-dissociated p24 anti- bination therapy. gen tests are highly specific for HIV infection and have been (3) Retest infants with initially negative virological tests at age used to diagnose infection in children. However, the use of p24 1–2 months. Using ZDV monotherapy to reduce perinatal 372 E. Mendelson et al. / Reproductive Toxicology 21 (2006) 350–382

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