CHẨN ĐOÁN NHIỄM TRÙNG BÀO THAI ,TORCHS

TRIỆU CHỨNG LÂM SÀNG, XÉT NGHIỆM CHẨN ĐOÁN NHIỄM TRÙNG BÀO THAI Ở TRẺ SƠ SINH

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 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 ∗ General introduction Rubella virus 2.1 Introduction 2.1.1 The pathogen 2.1.2 Immunity and protection 2.1.3 Laboratory assessment of primary rubella infection in pregnancy 2.1.4 Pre- and postnatal laboratory assessment of congenital rubella infection 2.2 Laboratory assays for assessment of rubella infection and immunity 2.2.1 Rubella neutralization test (NT) 2.2.2 Hemagglutination inhibition test (HI) 2.2.3 Rubella specific ELISA IgG 2.2.4 Rubella specific ELISA IgM 2.2.5 Rubella specific IgG-avidity assay 2.2.6 Rubella virus isolation in tissue culture Corresponding author Tel.: +972 530 2421; fax: +972 535 0436 E-mail address: ellamen@sheba.health.gov.il (E Mendelson) 0890-6238/$ – see front matter © 2006 Elsevier Inc All rights reserved doi:10.1016/j.reprotox.2006.02.001 352 352 352 352 352 355 357 358 358 358 358 358 359 359 E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 351 2.2.7 Rubella RT-PCR assay 2.3 Summary Cytomegalovirus (CMV) 3.1 Introduction 3.1.1 The pathogen 3.1.2 Laboratory assessment of CMV infection in pregnant women 3.1.3 Prenatal assessment of congenital CMV infection 3.2 Laboratory assays for assessment of CMV infection 3.2.1 CMV IgM assays 3.2.2 CMV IgG assays 3.2.3 CMV IgG avidity assays 3.2.4 CMV neutralization assays 3.2.5 Virus isolation in tissue culture 3.2.6 Detection of CMV by PCR 3.2.7 Quantitative PCR-based assays 3.3 Summary Varicella-zoster virus (VZV) 4.1 Introduction 4.1.1 The pathogen 4.1.2 Assessment of VZV infection in pregnancy 4.1.3 Prenatal and perinatal laboratory assessment of congenital VZV infection 4.2 Laboratory assays for assessment of VZV infection and immunity 4.2.1 VZV IgG assays 4.2.2 VZV IgM assays 4.2.3 Virus detection in clinical specimens 4.2.4 Virus isolation in tissue culture 4.2.5 Direct detection of VZV antigen 4.2.6 Molecular methods for detection of viral DNA 4.3 Summary Herpes simplex virus (HSV) 5.1 Introduction 5.1.1 The pathogen 5.1.2 Laboratory assessment of HSV infection in pregnancy and in neonates 5.2 Laboratory assays for assessment of HSV infection and immune status 5.2.1 HSV IgG assays 5.2.2 HSV type-specific IgG assays 5.2.3 HSV IgM assays 5.2.4 Virus isolation in tissue culture 5.2.5 Direct antigen detection of HSV 5.2.6 Detection of HSV DNA by PCR 5.3 Summary Parvovirus B19 6.1 Introduction 6.1.1 The pathogen 6.1.2 Laboratory assessment of parvovirus B19 infection in pregnancy 6.1.3 Prenatal laboratory assessment of congenital B19 infection 6.2 Laboratory assays for assessment of parvovirus B19 infection 6.2.1 B19 IgM and IgG assays 6.2.2 Detection of viral DNA in maternal and fetal specimens 6.2.3 Quantitative assays for detection of viral DNA 6.3 Summary Human immunodeficiency virus 7.1 Introduction 7.1.1 The pathogen 7.1.2 Importance of laboratory assessment of HIV infection in pregnancy 7.1.3 Prenatal laboratory assessment of HIV infection 7.2 Laboratory assessment of HIV infection 7.2.1 HIV antibody assays 7.2.2 Detection of viral DNA in maternal and newborn specimens 7.3 Summary Acknowledgments References 359 359 360 360 360 360 360 361 361 361 361 362 362 362 362 363 363 363 363 363 364 364 364 364 364 364 364 364 365 365 365 365 365 366 366 366 366 366 366 366 367 367 367 367 367 367 368 368 368 369 369 369 369 369 370 370 370 370 371 372 372 372 352 E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 General introduction Viral infections during pregnancy carry a risk for intrauterine transmission which may result in fetal damage The consequences of fetal infection depend on the virus type: for many common viral infections there is no risk for fetal damage, but some viruses are teratogenic while others cause fetal or neonatal diseases ranging in severity from mild and transient symptoms to a fatal disease In cases where infection during pregnancy prompts clinical decisions, laboratory diagnostic tests are an essential part of the clinical assessment process This review describes the six most important viruses for which laboratory assessement during pregnancy is required and experience has been gained over many years Rubella virus and CMV are teratogenic viruses, while VZV, HSV, parvovirus B19 and HIV cause fetal or neonatal transient or chronic disease The ability of viruses to cross the placenta, infect the fetus and cause damage depends, among other factors, on the mother’s immune status against the specific virus In general, primary infections during pregnancy are substantially more damaging than secondary infections or reactivations Laboratory testing of maternal immune status is required to diagnose infection and distinguish between primary and secondary infections Assessment of fetal damage and prognosis requires prenatal laboratory testing primarily in those cases where a clinical decision such as drug treatment, pregnancy termination or intrauterine IgG transfusion must be taken This review describes basic virological facts and explains the laboratory approaches and techniques used for the diagnostic process It aims at familiarizing physicians with the rational behind the laboratory requests for specific and timely specimens and with the interpretation of the tests results including its limitations The laboratory methods used for assessment of viral infections in general are of two categories: serology and virus detection Serology is very sensitive but often cannot conclusively determine the time of infection, which may be critical for risk assessment Traditional serological tests, which measure antibody levels without distinction between IgM and IgG, usually require two samples for determination of seroconversion or a substantial rise in titer The modern tests can distinguish between IgG and IgM and may allow diagnosis in one serum sample However, biological and technical difficulties are common and may cause false positive and false negative results The properties of all serological assays used for each of the viruses will be described in detail in the following chapters Virus detection is used primarily for prenatal diagnosis Invasive procedures must be used to obtain samples representing the fetus such as amniotic fluid (AF), cord blood and chorionic villi (CV) The traditional “gold standard” assay for virus detection used to be virus isolation in tissue culture, but other, more rapid and sensitive methods were developed in recent years Among the new methods are direct antigen detection by specific antibodies and amplification and detection of viral nucleic acids The general characteristics of all laboratory assays described in this article are summarized in Table Since the algorithm for maternal and fetal assessment and the interpretation of tests results vary from one virus to another, we have described the approach to each viral infection in a separate chapter Figs 1–6 depict the most common algorithms used for the laboratory diagnosis of each of the viral infections Rubella virus 2.1 Introduction 2.1.1 The pathogen Rubella is a highly transmissible childhood disease which can cause large outbreaks every few years It is a vaccine preventable disease and in developed countries outbreaks are mostly confined to unvaccinated communities [1] Rubella reinfection following natural infection is very rare Rubella virus (RV) is classified as a member of the togaviridae family and is the only virus of the genus rubivirus [2] Hemagglutinating activity and at least three antibody neutralization domains were assigned to the early proteins E1 and E2 [3–5] At least one weak neutralization domain was identified on E2 [5] The main route of postnatal virus transmission is by direct contact with nasopharyngial secretions [6] Postnatal RV infection is a generally mild and self-limited illness [6–8], but primary RV infections during the first trimester of pregnancy have high teratogenic potential leading to severe consequences, known as congenital rubella syndrome (CRS) which may occur in 80–85% of cases [8,9] It should be emphasized that more than 50% of RV infections in non-immunized persons in the general population (and in pregnant women) are subclinical [6–9] 2.1.2 Immunity and protection Antibody level of 10–15 international units (IU) of IgG per millilitre is considered protective Naturally acquired rubella generally confers lifelong and usually high degree of immunity against the disease for the majority of individuals [10,11] Rubella vaccination induces immunity that confers protection from viraemia in the vast majority of vaccinees, which usually persists for more than 16 years [10–12] A small fraction of the vaccinees fail to respond or develop low levels of detectable antibodies which may decline to undetectable levels within 5–8 years from vaccination [13–17] Several methods are used to determine immunity (Table 1) Neutralization test (NT) and hemagglutination inhibition test (HI) correlate well with protective immunity, but since they are difficult to perform and to standardize, they were replaced by the more rapid, facile and sensitive enzyme-linked immunosorbant assay (ELISA) [5,18] In our experience (unpublished data), there is a clear distinction between antibody levels measured using ELISA, and antibody levels measured using functional assays such as NT and HI Moreover, standardization of anti RV antibody assays using different techniques and a variety of antigens (i.e., whole virus, synthetic peptides, recombinant antigen, etc.) has not been achieved, leading to uncertainties regarding the antibody levels that confer immunity and protection against reinfections and against virus transmission to the 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 Summary and characteristics of the laboratory tests used for assessment of viral infections in pregnancy Laboratory test Serology Neutralization (NT) Hemagglutination inhibition (HI) ELISA IgM ELISA IgG IgG avidity (ELISA) Immunofluorescence (IFA; IFAMA, etc.) Western blot (WB) Virus detection Virus isolation in tissue culture Direct antigen detection Shell-vial assay Molecular assays PCR; RT-PCR Test principles Clinical samples Technical advantages Technical disadvantages limitations Interpretation of positive results Inhibition of virus growth in tissue culture by Aba Prevention of hemagglutination by binding of Ab to viral Agb Detection of virus specific Ab bound to a solid phase by a labled secondary anti-IgM Ab Detection of virus specific Ab bound to a solid phase by a labled secondary anti-IgG Ab Removal of low avidity IgG Ab which results in a reduced signal Maternal blood Corresponds with protection Done only in reference laboratories Maternal blood Accurate and corresponds with protection Laborious, not very sensitive, not Ab class-specific Laborious, not Ab class specific Used only for rubella, done only in specialized labs Neutralizing antibodies are present at a certain titer HI antibodies are present at a certain titer Maternal blood, fetal blood, newborn blood Fast and sensitive, commercialized, automated None False positive and false negative IgM antibodies are present Maternal blood, newborn blood Fast and sensitive, commercialized, automated None None IgG antibodies are present (sometimes with units) Maternal blood Fast and sensitive, commercialized, automated Not many available commercially No interpretation for results outside the inclusion or exclusion criteria Detection of IgG or IgM Ab which binds to a spot of virus infected cells on a slide by a labled secondary Ab Separated viral proteins attached to a nylon membrane react with patient’s serum and detected by labled anti-human Ab Maternal blood, fetal blood, newborn blood Can yield titer; short time Manual, reading is subjective Unsuitable for testing large numbers Low avidity: recent infection; medium avidity: not known; high avidity: probably old infection Antibodies are present at a certain titer Maternal blood infant’s blood Detects antibody specific to a viral protein Laborious Not very sensitive Antibodies specific to certain viral antigens are present Innoculation of specific tissue cultures with clinical samples and watching for CPEc Detection of a viral antigen in cells from a clinical sample by IFA or ELISA Any clinical sample which may contain virus Detects and isolates live virus Very labourious Slow Insensitive, done only in virology labs Live virus is present in the clinical sample For IFA: cells from clinical samples For ELISA: any sample Any clinical sample which may contain virus Fast and simple Not sensitive Not sensitive, low positive predictive value The sample most likely contains live virus Detects live virus; rapid: results within 16–72 h Labourious; requires high skills; uses expensive monoclonal Abs Not highly sensitive; done only in virology labs Live virus is present in the clinical sample Any clinical sample which may contain virus Fast, simple, can be automated; very sensitive Very prone to contaminations False positive by contamination; may detect latent virus Viral nucleic acid is present in the sample, not known if live virus is present Innoculation of specific tissue cultures with clinical samples, then fixation and detection of viral cell-bound antigen by IFA Enzymatic amplification of viral nucleic acid and detection of amplified sequences 354 E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 Table (Continued ) Laboratory test Real-time PCR/RT-PCR In situ hybridization In situ PCR a b c Test principles Clinical samples Technical advantages Technical disadvantages limitations Interpretation of positive results Detection of accumulating PCR products by a fluorescent dye or probe in a specialized instrument Detection of viral nucleic acid in smears or tissue sections by labled probes Detection of viral nucleic acid in smears or tissue sections by PCR using labled primers Any clinical sample which may contain virus Very fast, simple not prone to contaminations; can be quantitative Expensive instruments Sometimes too sensitive, interpretation of very low result questionable Viral nucleic acid is present in the sample (at a certain amount), not known if live virus is present Cells or tissue from clinical samples Sensitive and specific Difficult to perform Done only in specializing labs Viral nucleic acid is present in the sample, not known if live virus is present Cells or tissue from clinical samples Sensitive and specific Difficult to perform Doe only in specializing labs Viral nucleic acid is present in the sample, not known if live virus is present Antibody Antigen Cytopathic effect 50%) which were detected during an outbreak in Israel in 1992 occurred in the presence of low neutralizing antibody titers of 1:4 (cut off level), and sharp decline in the reinfection rate correlated with the presence of higher titers of neutralizing antibodies (unpublished data) Reinfection rates following vaccination are considerably higher than following natural infection, ranging between 10% and 20% [19] Many developed countries adopted the infant routine vaccination policy using MMR (mumps measles and rubella) vaccine designed to provide indirect protection of child-bearing age Fig 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 m) and on virus isolation from the newborn’s respiratory secretions E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 355 Fig 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 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 in other countries with high vaccination coverage, RV still circulates and may cause reinfections in vaccinated women whose immunity has waned [19,20,23, unpublished data] 2.1.3 Laboratory assessment of primary rubella infection in pregnancy Assessment of primary rubella infection in pregnant women relies primarily on the detection of specific maternal IgM antibodies in combination with either seroconversion or a >4-fold rise in rubella specific IgG antibody titer in paired serum samples (acute/convalescent) as shown in Fig Today, due to the high sensitivity of the ELISA-IgM assays low levels of rubella specific IgM are detected more frequently, leading to an increase in the number of therapeutic abortions and reducing the number of CRS cases However, frequently the low level of IgM detected is not indicative of a recent primary infection for several reasons: (a) IgM reactivity after vaccination or primary rubella infection may sometimes persist for up to several years [27–29]; (b) heterotypic IgM antibody reactivity may occur in patients recently infected with Epstein Barr virus (EBV), cytomegalovirus (CMV), human parvovirus B19 and other pathogens, leading to false positive rubella IgM results [30–35]; (c) false positive rubella specific IgM response may occur in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) or juvenile rheumatoid arthritis, etc., due to the presence of rheumatoid factor (RF) [36,37]; (d) low level of specific rubella IgM may occur in pregnancy due to Fig 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 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 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 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 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 [33,35,38] False negative results may also occur in samples taken too early during the course of primary infection Thus, the presence or absence of rubella specific IgM in an asymptomatic patient should be interpreted in accordance with other clinical and epidemiological information available and prenatal diagnosis may be required A novel assay developed recently to support maternal diagnosis is the IgG avidity assay (Table 1) which can differentiate between antibodies with high or low avidity (or affinity) to the antigen It is used when the mother has both IgM and IgG in the first serum collected (Fig 1) Following postnatal primary infection with rubella virus, the specific IgG avidity is initially low and matures slowly over weeks and months [39–41] Rubella specific IgG avidity measurement proved to be a useful tool for the differentiation between recent primary rubella (clinical and especially subclinical infection), reinfection, remote rubella infection or persistent IgM reactivity This distinction is critical for the clinical management of the case, since infection prompts a therapeutic abortion, reinfection requires fetal assessment, while remote infection or non-specific IgM reactivity carry no risk to the fetus [39–42] 2.1.4 Pre- and postnatal laboratory assessment of congenital rubella infection Maternal primary infection prompts testing for fetal infection (Fig 1) The preferred laboratory method for prenatal diagnosis is determination of IgM antibodies in fetal blood obtained by cordocentesis [27,43] Other options include virus detection in chorionic villi (CV) samples or amniotic fluid (AF) specimens The laboratory methods used for virus detection are virus isolation in tissue culture or amplification of viral nucleic acids by RT/PCR (Table 1) However, using those methods for detection of rubella virus in AF and CV might be unreliable, particularly in AF samples due to low viral load Studies showed that rubella virus may be present in the placenta but not in the fetus, or it can be present in the fetus but not in the placenta, leading to false negative results [6,43,44] Thus, according to one opinion, detection of rubella virus in AF or CV does not justify the risk of fetal loss following these invasive procedures [45], while according to another opinion, laboratory diagnosis of fetal infection should combine a serological assay (detection of rubella specific IgM) with a molecular method (viral RNA detection) in order to enhance the reliability of the diagnosis [46] A recent study showed 83–95% sensitivity and 100% specifity for detection of RV in AF by RT/PCR [47] Postnatal diagnosis of congenital rubella infection [9,27,36] is based on one or more of the following: a Isolation of rubella virus from the infant’s respiratory secretions b Demonstration of rubella specific IgM (or IgA) antibodies in cord blood or in neonatal serum, which remain detectable for 6–12 months of age c Persistence of anti-rubella IgG antibodies in the infant’s serum beyond 3–6 months of age The principles, advantages and disadvantages of each laboratory test, are described below 358 E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 2.2 Laboratory assays for assessment of rubella infection and immunity 2.2.1 Rubella neutralization test (NT) Virus neutralization is defined as the loss of infectivity due to reaction of a virus with specific antibody Neutralization can be used to identify virus isolates or, as in the case of rubella diagnosis, to measure the immune response to the virus [24,36,48] As a functional test, neutralization has proven to be highly sensitive, specific and reliable technique, but it can be performed only in virology laboratories which comprise only a small fraction of the laboratories performing rubella serology Rubella virus produces characteristic damage (cytopathic effect, CPE) in the RK-13 cell line that was found most sensitive and suitable for use in rubella neutralization test Other cells such as Vero and SIRC lines can be used if conditions are carefully controlled [36] Principally, 2-fold dilutions of each test serum are mixed and incubated with 100 infectious units of rubella virus under appropriate conditions Then cell monolayers are inoculated with each mixture and followed for CPE Control sera possessing known high and low neutralizing antibody levels and titrations of the virus are included in each test run The neutralization titer is taken as the reciprocal of the highest serum dilution showing complete inhibition of CPE [25,36] 2.2.2 Hemagglutination inhibition test (HI) Until recently, assessment of rubella immunity and diagnosis of rubella infection has been carried out mainly by the HI test which is based on the ability of rubella virus to agglutinate red blood cells [49] HI test is labor intensive, and is currently performed mainly by reference laboratories HI is the “gold standard” test against which almost all other rubella screening and diagnostic tests are measured During the test, the agglutination is inhibited by binding of specific antibodies to the viral agglutinin Titers are expressed as the highest dilution inhibiting hemagglutination under standardized testing conditions [50–53] The HI antibodies increase rapidly after RV infection since the test detects both, IgG and IgM class-specific antibodies A titer of l:8 is commonly considered negative (cut off level: 1:16) and a titer of ≥1:32 indicates an earlier RV infection or successful vaccination and immunity Seroconversion is interpreted as primary rubella infection, and a 4-fold increase in titer between two serum samples (paired sera) in the same test series, is interpreted as a recent primary rubella infection or reinfection [52] Considerable experience has been accumulated over the years in the interpretation of the clinical significance of HI titers [52–54], and the test results accurately correlate with clinical protection [5,18] Although HI is generally considered as not sensitive enough, in certain situations it is still in use for resolution of diagnostic uncertainties Detection of rubella specific IgM class antibodies by HI test which requires tedious methods for purification of IgM or removal of IgG [55,56], are no longer in use due to the development of a variety of rapid, easy to perform and sensitive methods, of which ELISA is the most vastly used [18,57] 2.2.3 Rubella specific ELISA IgG The ELISA technique was established for detection of an increasing range of antibodies to viral antigens In 1976, Voller et al [58] developed an indirect assay for the detection of antiviral antibodies The technique has been successfully applied for the detection of rubella specific antibodies Almost all commercially available ELISA kits for the detection of rubella specific IgG are of the indirect type, employing rubella antigen attached to a solid phase (microtiter polystyrene plates or plastic beads) The source of the antigen (peptide, recombinant or whole virus antigen) affects the sensitivity and specificity of the assay After washing and removal of unbound antigen, diluted test serum is added and incubated with the immobilized antigen The rubella specific antibodies present in the serum bind to the antigen Then, unbound antibodies are removed by washing and an enzyme conjugated anti-human IgG is added and further incubation is carried out The quantity of the conjugate that binds to each well is proportional to the concentration of the rubella specific antibodies present in the patient’s serum The plates are then washed and substrate is added resulting in color development The enzymatic reaction is stopped after a short incubation period, and optical density (OD) is measured by an ELISA-reader instrument The test principle allows the detection of IgM as well by using an appropriate anti-human IgM conjugate [53,57] In most commercial ELISA IgG assays the results are automatically calculated and expressed quantitatively in international units (IU) When performed manually, the procedure takes approximately h but automation has reduced it to about 30 [57–59] It is important to note that in order to obtain reliable results, determination of a significant change in specific IgG activity in paired serum samples should always be performed in the same test run and in the same test dilution The correlation between the ELISA and HI or NT titers is not always high This may be explained by the fact that the three methods detect antibodies directed to different antigenic determinants [54] Certain individuals fail to develop antibodies directed to protective epitopes such as the neutralizing domains of E1 and E2 due to a defect in their rubella specific immune responses [21] but they develop antibodies directed to antigenic sub-regions of rubella virus proteins ELISA assays utilizing whole virus as antigen may fail to distinguish between these different antibody specificities Thus, seroconversion determined by ELISA based on a whole virus antigen does not necessarily correlate with protection against infection [52] 2.2.4 Rubella specific ELISA IgM Commercially available ELISA kits for the detection of IgM are mainly of two types: a Indirect ELISA: The principle of the assay was described above for rubella IgG except for using enzyme labeled antihuman IgM as a conjugate In this assay, false negative results may occur due to a competition in the assay between specific IgG antibodies with high affinity (interfering IgG) while the specific IgM have lower affinity for the antigen [31,32] In the E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 new generation ELISA assays this is avoided by the addition of an absorbent reagent for the removal of IgG from the test serum False positive results may occur if rheumatoid factor (RF: IgM anti-IgG antibodies) is present along with specific IgG in the test serum Absorption or removal of RF and/or IgG is necessary prior to the assay to avoid such reactions [30–32,60] b IgM capture ELISA: In these assays anti-human IgM antibody is attached to the solid phase for capture of serum IgM Rubella virus antigen conjugated to enzyme-labeled anti-rubella virus antibody is added for detection This type of assay eliminates the need for sample pretreatment prior to the assay [32,61] As for the rubella virus antigens, most assays are based on whole virus extracts, but recent developments led to production of recombinant and synthetic rubella virus proteins [5,62] 2.2.5 Rubella specific IgG-avidity assay This assay is based on the ELISA IgG technique and applies the elution principle in which protein denaturant, mostly urea (but also diethylamine, ammonium thiocyanate, guanidine hydrochloride, etc.) is added after binding of the patient’s serum The denaturant disrupts hydrophobic bonds between antibody and antigen, and thus, low avidity IgG antibodies produced during the early stage of infection are removed This results in a significant reduction in the IgG absorbance level [63] The avidity index (AI) is calculated according to the following formula [57]: absorbance of avidity ELISA absorbance of standard ELISA The AI is a useful measure only when the IgG concentration in the patient’s serum is not below 25 IU [39] Low avidity (usually below 50%) is associated with recent primary rubella infection while reinfection is typically associated with high avidity as a result of the stimulation of memory B cells (immunological memory) [39–41] In infants with CRS the low avidity IgG continues to be produced for much longer than in cases of postnatal primary rubella, where it lasts 4–6 week after exposure [39] This may be used for retrospective assessment of initially undiagnosed CRS cases AI = 100 × 2.2.6 Rubella virus isolation in tissue culture Diagnosis of prenatal or postnatal rubella infections are essentially based on the more reliable and rapid serological techniques However, virus isolation is useful in confirming the diagnosis of CRS (Fig 1) and rubella virus strain characterization required for epidemiological purposes Rubella virus can be isolated using a variety of clinical specimens such as: respiratory secretions (nasopharyngeal swabs), urine, heparinized blood, CSF, cataract material, lens fluid, amniotic fluid, synovial fluid and products of conception (fetal tissues: placenta, liver, skin, etc.) obtained following spontaneous or therapeutic abortion [6,36,44,64] In order to avoid virus inactivation, specimens should be inoculated into cell culture immediately or stored at ◦ C for not more than days, or kept frozen (−70◦ C) for longer periods [36] 359 Rubella virus can be grown in a variety of primary cells and cell lines [36,65], but RK-13 and Vero cell lines are the most sensitive and suitable for routine use In these cell systems rubella virus produces characteristic CPE Since the CPE is not always clear upon primary isolation, at least two successive subpassages are required [66] When CPE is evident the identity of the virus isolates should be confirmed using immunological or other methods [36,65,67] 2.2.7 Rubella RT-PCR assay Reverse transcription followed by PCR amplification (RTPCR) is a rapid, sensitive and specific technique for detection of rubella virus RNA in clinical samples using primers from the envelope glycoprotein E1 open reading frame [45,46,68,69] Coding sequences for a major group of antigenic determinants are located between nucleotides 731 and 854 of the E1 gene of RV strain M33 This region is highly conserved in various wild type strains and is likely to be present in most clinical samples from rubella infected patients Specific oligonucleotide primers located in this region were designed for amplification by RT-PCR [70–72] Following rubella genomic RNA extraction from clinical specimens and RT-PCR amplification, the product is visualized by gel electrophoresis Positive samples show a specific band of the expected size compared to size markers [68,69,72] A nested RT-PCR assay, in which the RT-PCR product from the first amplification reaction is re-amplified by internal primers, was developed and shown to provide a higher level of sensitivity for the detection of rubella virus RNA [72] However, the risk of contamination is markedly increased The detection limit of the RT-PCR assay is approximately two RNA copies Clinical specimens for rubella virus genome detection include: products of conception (POC), CV, lens aspirate/biopsy, AF, fetal blood, pharyngeal swabs and spinal fluid (CSF) or brain biopsy when the central nervous system (CNS) is involved [68,69,73–75] An additional advantage of RT-PCR is that it does not require infectious virus [74] RV is extremely thermo-labile and frequently is inactivated during sample transporation to the laboratory Finally, it should be noted that clinical samples may contain PCR inhibitors (such as heparin and hemoglobin), and the extraction procedure itself may cause enzyme inhibition [72,76,77] This underscores the need and importance for strict internal quality control during each step of the RT-PCR procedure and participation in external quality assessment programs is of a high value 2.3 Summary Rubella infection during pregnancy, although rare in countries with routine vaccination programs, is still a problem requiring careful laboratory assessment The laboratory testing should confirm or rule-out recent rubella infection in pregnant women and identify congenital rubella infections in the fetus or neonate Maternal infection is currently assessed by serological assays, primarily by ELISA IgM and IgG Borderline results for the IgG assay can be further assessed by the HI or NT assays available 368 E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 does not always develop IgM it is necessary to detect the virus itself in fetal samples B19 cannot be cultivated and therefore detection of viral DNA is sought The type of specimen and the detection methods are still controversial: fetal blood and AF are the most common specimens obtained and the most highly sensitive molecular methods are employed However, 10–25% of the specimens from asymptomatic fetuses may have B19 DNA [289,290] This drives the need to develop quantitative molecular assays which can determine viral load and possibly associate it with the risk to the fetus Those assays include quantitative PCR-ELISA, in situ hybridization and rt-PCR The latter has advantages which will be discussed below [304–308] 6.2 Laboratory assays for assessment of parvovirus B19 infection 6.2.1 B19 IgM and IgG assays Several commercial tests have been developed on the basis of recombinant and synthetic antigens [309–311] These recombinant antigens frequently lack important conformational epitops reducing the sensitivity and specificity of the assays Moreover, “gold standard” methods like virus NT or HI assays are absent, and it is difficult to assess the sensitivity and specificity of the serological assays The variation in specificity and sensitivity among commercial assays is high, and discordant results are often obtained indicating false positive and false negative responses [314–317] Studies aimed at evaluating the sensitivity and specificity of various such assays were mostly based on comparative evaluation, using samples selected on the basis of clinical diagnosis In a study conducted in Norway [315], five commercial ELISA and IF IgM kits were comparatively evaluated in three groups of patients The calculated specificities for the kits were 70.1–94.8%, but the authors refrained from determining sensitivity because of the absence of suitable reference methods In another study conducted in Sweden [316], four commercial IgM assays were evaluated in comparison to an IgM antibody capture radioimmunoassay as a reference method [318] The calculated sensitivity varied between 90% and 97% and the specificities varied from 88% to 96% In a third study conducted in the USA [314] three ELISA systems utilizing one or more conformational antigens for detection of B19 IgM or IgG in sera of 198 pregnant women were comparatively evaluated Agreement with the consensus results varied from 92.3% to 100% for IgM and from 97.9% to 99.5% for IgG The authors note the high agreement in this study compared to their previous study [312] relating to the fact that the antigens utilized in the two studies were substantially different: the earlier study used a linear VP1 antigen while the latter one used a conformational VP2 antigen A study designed to evaluate the most appropriate assays for IgG detection was conducted in Italy [313] which compared the performance of three commercial assays using different antigens: (a) an ELISA assay using VP1 + VP2 recombinant native conformational antigens, (b) an ELISA assay using VP2 recombinant native conformational epitopes, (c) a Western blot assay using denatured linear antigens Four hundred and fourty six serum samples from blood donors with no IgM were tested Overall, 353 sera were found positive by all methods combined Of those 98.6%, 94.6% and 89% were positive by assays a, b and c, respectively Some sera reacted only with conformational epitops The results of this study underscore the need to use ELISA-IgG assays which include conformational epitopes of both VP1 and VP2 or VP2 alone, and demonstrate the lower sensitivity of the Western blot assay In conclusion, when evaluating recent or past infections in pregnant women it is important to use well-established ELISA assays, and in cases of discrepancy between the test results and the clinical and epidemiological circumstances, confirmation must be sought using other commercial assays as well as supportive molecular assays discussed next 6.2.2 Detection of viral DNA in maternal and fetal specimens Due to the limitations of the serological assays described above and since viremia is long-lasting in B19 infections, molecular assays for detection of B19 viral DNA were developed early The methods most frequently used are DNA hybridization and PCR Molecular hybridization assays employing DNA probes derived from most of the viral genome are labeled with 32 P, biotin or digoxigenin [319–322] and are capable of detecting approximately 104 –105 genome copies/ml [275,323] This assay is sensitive enough to detect viral DNA in serum during peak viremia since the viral load exceeds 1010 genome copies/ml, but low levels of viremia may be missed [275,296,298] The PCR assays, developed later relied on a variety of primer sets derived from different genomic regions and were capable of detecting 102 –105 genome copies/ml [324,325] The nested PCR, the PCR-ELISA and the PCR-hybridization assays increased sensitivity and moved the detection limit down to 1–10 genome copies/ml [291,292,296,297,299,326–328] By using those highly sensitive methods it was revealed that in many cases serology failed to detect maternal or fetal infections, as some seronegative mothers as well as fetuses turned out positive in the DNA detection assays This finding was not surprising in view of the known limitations of the serological assays The ability to detect maternal and fetal infection by the highly sensitive molecular methods allowed correct diagnosis of previousely unresolved cases, particularly cases of intrauterine infection which did not result in fetal hydrops or were completely subclinical [275,276,290,292,296,297,324,327,329] However, as stated above, low levels of B19 DNA in maternal blood can be unrelated to recent infection The use of various techniques without standardization, the epidemiologically variable circumstances (epidemic VS nonepidemic years), and the use of different primer sets and clinical specimens led to a high variability in the sensitivity, specificity and interpretation of the assays results Moreover, new genotypes discovered recently were missed by common primer sets Comparison of IgM and DNA detection by PCR in fetal blood was conducted in a study done on 57 pregnant women and their fetuses who had abnormal ultrasonography [327] Viral DNA was found by PCR in 16 out of 58 fetuses (27%) while IgM was E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 detected only in (12.3%) Two fetuses had false-positive IgM result, not supported by any other findings Other researchers [276] compared maternal IgM to fetal serum or AF PCR results in 56 women at high risk for B19 infection They found positive PCR in 24 IgM-negative/IgG-positive and seronegative (total 50%) out of, in addition to positive PCR in 15 (26%) of IgM-positive women Another group [282] reported detection of viral DNA in fetal serum or AF by a PCR-hybridization assay in 11 out of 80 cases of fetal hydrops (14%) while maternal IgM antibodies were detected only in (3.7%) Finally, a comprehensice prospective study in 18 fetal hydrops cases conducted in Italy [275] examined maternal serum, fetal cord blood and amniotic fluid using nested PCR, dot-blot hybridization and in situ hybridization (ISH) The results showed that the ISH assay in fetal blood cells was 100% sensitive while the other methods missed few to many cases The conclusion drawn from this study is that various assays have complementary roles, and reliable diagnosis can be achieved only by a combination of serological and molecular assays done on maternal and fetal samples as outlined in Fig 6.2.3 Quantitative assays for detection of viral DNA The need to determine the quantity of B19 viral DNA present in clinical samples (viral load) arose because B19 can establish long-lasting persistent infection in immunocompetent individuals It is not clear if low viral loads reflect whole genomes or viable infectious virus particles, or only pieces of viral DNA Studies in seronegative plasma-pool recipients showed that only recipients of plasma containing >107 genome copies/ml became infected or seroconverted [305] B19 DNA can also be detected 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 the absence of any detectable congenital abnormalities, and the outcome of detectable fetal infection, including fetal hydrops, varies from spontaneous resolution to still-birth Real-time PCR assays developed recently are equivalent to or more sensitive than nested PCR and PCR-ELISA but are much less prone to molecular contamination and produce a quantitative result which can be standardized and automated [304,306–308,332] In a retrospective study, Knoll et al [332] used real-time PCR to investigate the viral load in paired samples from mothers and their abnormal fetuses, and from mothers with normal fetuses who were exposed to B19 The viral load in the maternal serum ranged from 7.2 × 102 to 2.6 × 103 The authors did not report statistically significant correlation between maternal or fetal viral load and fetal condition However, they noted that they could not exclude a correlation between peak maternal viremia levels and fetal condition since most of the mothers were not aware of their infection until onset of fetal symptoms, and their serum was collected after peak viremia Although the viral load in fetal sera was higher than in AF samples, the rtPCR assay detected all positive cases using either one of these fetal specimens The authors recommend testing AF rather than fetal blood because AF can be drawn earlier, is simpler to obtain and less risky to the fetus Clearly, more prospective studies are necessary on larger groups of patients to learn more about the association between viral load and pregnancy outcome 369 6.3 Summary Parvovirus B19 infection during pregnancy may cause fetal damage resulting in fetal loss Early diagnosis of maternal infection will allow fetal assessment and treatment by intrauterine blood transfusion Unfortunately, mothers often are not aware of their infection until fetal damage is observed Confirmation of B19 infection requires laboratory assessment, which is complicated by the nature of the viral infection and immune response Serology, performed by using ELISA assays rely on recombinant antigens and concordance is low among all commercial assays available In the absence of a “gold standard” assay false positive and false negative results prevail Virus culturing is impossible and virus detection is based on various molecular assays In spite of several studies there is no consensus regarding the most appropriate clinical specimen and method for detection of viral DNA Currently, on practical grounds, it is recommended to use ELISA IgM and IgG assays based on recombinant conformational epitopes of VP1 and VP2 or VP2 alone, and to use AF or fetal serum for detection of fetal infection by the most sensitive molecular methds available (nested PCR or rt-PCR) Since B19 may establish long lasting infection in the absence of symptoms, interpretation of viral DNA detection in maternal blood is difficult Assessment of fetal infection and risk should rely on the clinical situation and other prenatal diagnostic means An algorithm describing the most practical approach to laboratory assessment of B19 infection in pregnancy is shown in Fig Human immunodeficiency virus 7.1 Introduction 7.1.1 The pathogen Human immunodeficiency virus (HIV) is a retrovirus that infects helper T cells of the immune system causing a progressive reduction in their numbers, and eventually acquired immunodeficiency syndrome (AIDS) HIV is a member of the Retroviridae family [333], genus Lentivirus (or “slow” viruses) The course of infection with these viruses is characterized by a long interval between initial infection and the onset of serious symptoms The single-stranded RNA viruses exploit their reverse transcriptase enzyme to synthesize DNA using their RNA as a template The DNA is then incorporated into the genome of infected cells AIDS was first diagnosed in European sailors with African connections [334–336] The first AIDS virus, HIV-1, was initially identified in 1983 [337–341] A second AIDS virus, HIV-2, was discovered in 1986 [342–344] Forty million people were estimated to be infected with HIV at the end of 2004 [345] The highest prevalence is found in Sub-Saharan Africa and it is rising mainly in Asia and some of the former Soviet Union countries like Ukraine and the Russian Federation [345,346] During its spread among humans, group M HIV-1 (one of three groups: M, O and M), has evolved into multiple subtypes that differ from 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 sexual intercourse (both homosexual and heterosexual), sharing of needles by IV drug users, medical procedures using HIV-contaminated blood, tissues or equipment, and motherto-child transmission (MTCT) The likelihood of transmission is increased by factors that may damage mucosal linings of exposed tissues, especially by other sexually transmitted diseases that cause ulcers or inflammation HIV can be transmitted from infected mothers to infants during pregnancy (intrauterine) through transplacental passage of the virus [351], during labor and delivery (intrapartum) through exposure to infected maternal fluids (blood or vaginal secretions) [352–355] and during the post partum period through breastfeeding [356–359] Infants who have a positive virologic test (see below) at or before age 48 h are considered to have early (i.e., intrauterine) infection, whereas infants who have a negative virologic test during the first week of life and subsequent positive tests are considered to have late (i.e., intrapartum) infection [360] In the absence of breast-feeding, intrauterine transmission accounts for 10–35% of infection, and 60–75% of transmission occurs during labor and delivery [361–363] Without intervention, maternal infection leads to about 25–30% of babies being infected [364–369] Use of anti-retroviral therapy (ART) during pregnancy in HIV-infected women is associated with improved obstetric outcome of reduced infection rates of babies to less than 2% [370–373] and little maternal toxicity [374–376] The current US guidelines are to offer all pregnant HIV-1-infected women highly active antiretroviral therapy (HAART) to maximally suppress viral replication, reduce the risk of prenatal transmission, and minimize the risk of development of resistant virus In addition, HIV-infected women are offered an elective caesarean section delivery [355] The results also suggest that special attention should be given to women belonging to previously identified risk groups Because testing has proven very successful in helping to prevent the spread of the disease to babies, a US federal panel has recommended that all pregnant women, not just those considered at high risk, be screened for the AIDS virus [377–381] No prenatal (intrauterine) diagnosis is performed because: (a) the infection can occur during delivery and (b) the testing intervention may increase the chance of virus transmission to the baby Assessing the infection status of the mother is critical [381] and following delivery the new-born should be tested The laboratory testing is essential for the diagnosis 7.1.2 Importance of laboratory assessment of HIV infection in pregnancy Identification of HIV-infected women before or during pregnancy is critical to providing optimal therapy for both infected women and their children and to preventing perinatal transmission (see below) For women with unknown HIV status during active labor, ART can still be effective when given during labor and delivery, followed by treatment of the newborn [382] This expedited intervention requires the use of rapid diagnostic testing during labor or rapid return of results from standard testing Knowledge of maternal HIV infection during the antenatal period enables HIV-infected women to receive appropriate antiretroviral therapy during pregnancy, during labor, and to newborns to reduce the risk for HIV transmission from mother to child [370,383,384] It also allows counseling of infected women about the risks for HIV transmission through breast milk and advising against breast feeding in countries where safe alternatives to breast milk are available [385] Early diagnostic evaluation of HIV-exposed infants permits early initiation of aggressive antiretroviral therapy in infected infants and initiation of prophylaxis against Pneumocystis carinii pneumonia (PCP) in all HIV-exposed infants beginning at age 4–6 weeks in accordance with Public Health Services (PHS) guidelines [386] 7.1.3 Prenatal laboratory assessment of HIV infection Prenatal laboratory assessment of congenital HIV infection in AF or cord blood is not recommended as the invasive procedures increases the risk of transmitting the virus from the maternal to the fetal blood stream 7.2 Laboratory assessment of HIV infection 7.2.1 HIV antibody assays The initial screening for HIV infection in adults and children is done by testing for antibodies Viral load assays are not intended for routine diagnosis but could be used in clinical management of HIV-infected persons in conjunction with clinical signs and symptoms and other laboratory markers of disease progression Detection of HIV-1 p24 antigen (Table 1) is used for routine screening in blood and plasma centers but their routine use for diagnosing HIV infection in individuals has been discouraged because the estimated average time from detection of p24 antigen to detection of HIV antibody by standard enzyme-immuno-assay (EIA) is days, and not all recently infected persons have detectable levels of p24 antigen [387] In the USA several FDA approved tests are available that enable the testing of HIV antibodies in different body fluids such as whole blood, serum, plasma, oral fluid and urine The standard testing algorithm for HIV-1 consists of initial screening with an EIA to detect antibodies to the virus Reactive specimens undergo confirmatory testing with a more specific supplemental test, usually a Western blot assay (WB) or, less commonly, IFA (Table 1) [388] Using both tests increases accuracy of the results while maintaining their sensitivity [389–391] Only specimens that are repeatedly reactive by EIA and positive by IFA or reactive by WB are considered HIV-positive and indicative of HIV infection [389,390,392,393] Incomplete antibody responses that produce negative or indeterminate results on WB tests can occur among persons recently infected with HIV who have low levels of detectable antibodies (i.e., seroconverting), persons who have end-stage HIV disease, and perinatally exposed but uninfected infants who are seroreverting (i.e., losing maternal antibody) Non-specific reactions producing indeterminate results in uninfected persons have occurred more frequently among pregnant women than among other persons [389–391,394] False-positive WB results are rare [395] E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 7.2.2 Detection of viral DNA in maternal and newborn specimens 7.2.2.1 Diagnosis of HIV infection in maternal specimens Pregnant women are screened for the presence of antibodies using the standard testing algorithm for adults (EIA plus WB or IFA) as shown in Fig For women with unknown HIV status during active labor, rapid diagnostic tests are used if rapid return of results from standard testing is not available 7.2.2.2 Diagnosis of HIV infection in newborns Infant HIV testing should be done as soon after birth as possible so appropriate treatment interventions can be implemented quickly [377,384] The standard antibody assays used for older children and adults are not useful for diagnosing children younger than 18 months as the presence of maternal antibodies makes serologic tests uninformative Therefore, a definitive diagnosis of HIV infection in early infancy requires viral diagnostic assays, including HIV-1 p24 antigen assays, nucleic acid amplification (e.g., PCR) or viral culture HIV infection can be definitively diagnosed in most infected infants by age month and in virtually all infected infants by age months HIV infection is diagnosed by at least two positive assays using two separate specimens [378] HIV DNA PCR is the preferred virologic method for diagnosing HIV infection during infancy It has 99% specificity to identify HIV proviral DNA in peripheral blood mononuclear cells (PBMC) obtained from whole blood samples collected in EDTA-containing tubes [396] A meta-analysis of published data from 271 infected children indicated that HIV DNA PCR was sensitive for the diagnosis of HIV infection during the neonatal period Thirty-eight percent of infected children had positive HIV DNA PCR tests by age 48 h, 93% by age 14 days and 96% by age 28 days No substantial change in sensitivity during the first week of life was observed, but sensitivity increased rapidly during the second week Quantitative assays that detect HIV RNA in plasma (see Section 7.2.2.3 below) appear to be as sensitive as HIV DNA PCR for early diagnosis of HIV infection in HIV-exposed infants [397–402] The specificity is comparable between the two tests, but results of HIV RNA load below 104 copies/ml should be interpreted with caution [403] Some clinicians use HIV RNA assay as the confirmatory test for infants testing HIV DNA PCR positive since it provides viral load measurement which guides treatment decisions Available quantitative RNA tests include the Amplicor HIV-1 monitor test 1.5 (Roche Diagnostics), the NASBA EasyQ HIV-1 (BioMerieux), the Quantiplex HIV RNA 3.0 (bDNA) (Bayer) and the LCx HIV RNA quantitative assay (Abbott Laboratories) assays [416–421] However, special attention should be taken where non-B HIV is expected HIV culture for the diagnosis of infection has a sensitivity that is similar to that of HIV DNA PCR [404] However, HIV culture is more complex and expensive to perform than DNA PCR, and definitive results may not be available for 2–4 weeks Both standard and immune-complex-dissociated p24 antigen tests are highly specific for HIV infection and have been used to diagnose infection in children However, the use of p24 371 antigen testing alone is not recommended because of its substantiallty reduced sensitivity and specificity compromising the critical need for timely diagnosis [405] Whether the current, more intensive antiretroviral combination regimens women may receive during pregnancy for treatment of their own HIV infection will affect diagnostic test sensitivity in their infants is unknown Similarly, if more complex regimens are administered to HIV-exposed infants for perinatal prophylaxis, the sensitivity of diagnostic assays will need to be re-examined [401] 7.2.2.3 Different subtypes HIV subtype B is the predominant viral subtype found in the U.S and western Europe Non-subtype B viruses predominate in other parts of the world, such as subtype C in regions of Africa and India and subtype E in much of southeast Asia Currently the available HIV DNA PCR commercial tests are less sensitive for detection for non-subtype B HIV, and false negative HIV DNA PCR assays have been reported in infants infected with non-subtype B HIV [406–410] Caution should be exercised in the interpretation of negative HIV DNA PCR test results in infants born to mothers who may have acquired an HIV non-B subtype Some of the currently available HIV RNA assays have improved sensitivity for detection of non-subtype B HIV infection [411–413], although even these assays may not detect some non-B subtypes, particularly group O HIV strains [414] In cases of infants where non-subtype B perinatal exposure may be suspected and HIV DNA PCR is negative, repeat testing using one of the newer RNA assays shown to be more sensitive for non-subtype B HIV is recommended (for example, the Amplicor HIV-1 monitor test 1.5, Nuclisens HIV-1 qt or Quantiplex HIV RNA 3.0 (bDNA) assays) In children with negative HIV DNA PCR and RNA assays but in whom non-subtype B infection continues to be suspected, the clinician should consult with an expert in pediatric HIV infection and the child should undergo close clinical monitoring and definitive HIV serologic testing at 18 months of age 7.2.2.4 Test algorithm for neonates HIV infection is diagnosed by two virological tests performed on separate blood samples, regardless of age The testing rules are summarized below and shown in Fig 6: (1) Initial testing is recommended by age 48 h As many as 40% of infected infants can be identified at this time Blood samples from the umbilical cord should not be used for diagnostic evaluations, because of concerns regarding potential contamination with maternal blood (2) Repeated diagnostic testing can also be considered at age 14 days in infants with negative tests at birth The diagnostic sensitivity of virological assays increases rapidly by age weeks Early identification of infection would permit discontinuation of neonatal ZDV chemoprophylaxis and a further evaluation of the need for more aggressive drug combination therapy (3) Retest infants with initially negative virological tests at age 1–2 months Using ZDV monotherapy to reduce perinatal 372 (4) (5) (6) (7) (8) (9) E Mendelson et al / Reproductive Toxicology 21 (2006) 350–382 transmission did not delay the detection of HIV in infants in PACTG protocol 076 [370,400–402,415] At age 3–6 months retest HIV-exposed children who have had repeatedly negative virological assays at birth and at age 1–2 months HIV infection can be reasonably excluded in non-breast fed infants with two or more negative virologic tests performed at age >1 month, with one of those being performed at age >4 months [386] Two or more negative HIV immunoglobulin G (IgG) antibody tests performed at age >6 months with an interval of at least month between the tests can also be used to reasonably exclude HIV infection in HIV-exposed children with no clinical or virologic laboratory evidence of HIV infection Serology after 12 months is recommended to confirm that maternal HIV antibodies transferred to the infant in utero have disappeared if there has not been previous confirmation of two negative antibody tests If the child is still antibody-positive at 12 months, then testing should be repeated between 15 and 18 months [387] Loss of HIV antibody in a child with previously negative HIV DNA PCR tests definitively confirms that the child is HIV uninfected A positive HIV antibody test at >18 months of age indicates HIV infection [378] 7.3 Summary Identification of HIV-infected women before or during pregnancy is critical to providing optimal therapy for both infected women and their children and to preventing perinatal transmission Pregnant women are screened for the presence of antibodies using the standard testing algorithm for adults (EIA plus WB or IFA) Prenatal laboratory assessment of congenital HIV infection is not recommended as it increases the risk of infecting the fetus, but extensive testing is performed to assess the infection status of the baby following delivery A definitive diagnosis of HIV infection in early infancy requires repeated testing using virological assays, with HIV DNA PCR being the currently preferred method All infected infants can be definitively diagnosed by age months An algorithm describing the laboratory diagnostic assays for HIV infection in pregnant women and neonates is shown in Fig Acknowledgments We are grateful to Galit Zemel for her extensive technical support in organizing the citations and reference list and in preparing the review for submission We also thank Zehava Yosefi for helping in reference retrieval and typing References [1] Hahne SJ, Abbink F, van Binnendijk RS, Ruijs WL, van Steenbergen JE, de Melker HE [Rubella epidemic in the Netherlands 2004/’05: awareness of congenital rubella syndrome required] Ned Tijdschr Geneeskd 2005;149(21):1174–8 [2] Melnick JL Taxonomy of viruses Prog Med Virol 1976;22:211– 21 [3] Oker-Blom C, Kalkkinen N, Kaariainen L, Pettersson RF Rubella 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