I. Myths about TORCH screening Question 1: Can TORCH screening diagnose birth defects? No. TORCH screening is the diagnosis of infection in pregnant women, which provides concern for the presence of infection and developmental defects in the fetus. TORCH screening screens individuals (infected) at risk for a certain disease (virus) in the population (pregnant women), diagnoses the latter (diagnosis of fetal infection), and intervenes with patients (fetuses) or carriers of the disease (virus) (pregnant women) for prevention and treatment purposes. Question 2: What are the common features of TORCH infection? 1. Mother-to-child transmission, fetal risk in T1 and neonatal risk in T3. 2. Pregnant women are asymptomatic or have very mild symptoms. 3, The virus can cause intrauterine infection through the placenta, which can cause premature birth, miscarriage, stillbirth or malformation. 4, the virus through the birth canal or breast milk infection of newborns cause neonatal multi-system and organ damage, mental retardation. 5, pregnant women are infected, the fetus is not necessarily infected, and fetal infection does not necessarily cause birth defects. Question 3: What are the differences of TORCH infection? The organism is infected with Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus serological changes vary, as do antibody changes, and quantitative testing of the degree of antibody changes is needed to make a correct judgment. Question 4: What are the different clinical significance of the indicators for TORCH infection testing? 1, direct indicators (viral antigen, viral DNA, viral RNA viral culture) detection is the virus itself, related to the replication pattern and latent location of the virus and other characteristics, suitable for diagnosis. 2. Indirect indicators (IgG, IgM) detect the immune response generated by the body after the virus stimulates the body, which is related to the immune function of the individual and is suitable for screening and immune status assessment. Question 5: How many types of infections are there in pregnancy? Infections during pregnancy are divided into initial (primary) infections, previous infections, recurrent infections, and reinfections, the concepts of which should not be confused. 1. Initial infection, also known as primary infection: the first time a pregnant woman’s serum is positive for virus-specific antibodies IgG and the previous serological test is negative is called initial infection. Only if a previous screening test has been done and the result is negative and archived (or the pregnant woman’s serum specimen is preserved), doing so will determine the initial infection. IgG antibody affinity assay in case of positive IgG helps to distinguish whether the infection is an acute initial infection, a previous infection or a recurrent infection and to estimate the time of initial infection, i.e. if the test result is high affinity, it can be concluded that the time of initial infection was 3 months ago. 2.Previous infection: the virus has been infected before and the body has produced antibodies or the virus is dormant and exists in a latent state. 3, recurrent infection: intermittent excretion of the virus in the presence of host immune function, is the latent state of endogenous virus reactivation. 4, reinfection: an individual who has been immunized is exposed to an exogenous new virus and reinfection occurs. It is not possible to distinguish recurrent infection and reinfection by serological methods, but only by viral isolation and molecular biological methods. 5. Congenital infection: the result of transplacental transmission of the virus. Initial or recurrent infection of the mother can transmit the virus to the fetus, resulting in congenital infection of the fetus. Question 6: Why should screening test IgG and IgM be done at the same time? Screening tests for IgG and IgM should be done at the same time, as IgM alone often gives incorrect results. positive IgM is not sufficient proof of recent infection, and in some populations IgM can be present continuously for several years after infection, so a positive IgM alone is not diagnostic. For example, the IgM of non-acute infection with rubella is an example. The reasons for this can be divided into the following two situations: 1. True positive IgM: This is due to the fact that some people continue to have IgM expression in their bodies for many years after the occurrence of infection, and often the IgM level remains low and stable, often accompanied by a positive IgG and also remains stable. At this time, the IgM results obtained by the kit are correct, but it does not mean that the individual is experiencing acute infection, at this time, if only IgM is detected alone, it will cause misjudgment. 2, IgM false positive: this is mainly due to rheumatoid factor (RF), cross-reactivity or polyclonal stimulation and other factors that lead to the wrong results of the kit, which is due to the inherent limitations of the immunological detection means itself, can not be completely avoided. Positive IgM results for non-acute infections due to the above two conditions can then cause confusion in clinical diagnosis, as these individuals are not undergoing acute infection. However, this problem can be satisfactorily corrected by concurrent IgG testing with quantitative detection reagents. In cases where the initial test is IgM-positive/IgG-negative, the second test 15 days-1 month later, the individual will remain so or IgM will turn negative, i.e., no serological conversion of IgG will occur; if the initial test is IgM-positive or /IgG-positive, the second test 15 days-1 month later will tend to show no significant change in the values of these two indicators. In particular, IgG will remain stable because the individual has not experienced a true recurrence of infection. Question 7: Why is TORCH screening in pregnancy time-sensitive? Different viruses have different screening goals and different requirements for the week of gestation, and without a week of gestation, screening results are often meaningless. Some viruses are meaningful when screened in early pregnancy and some are meaningful when screened in late pregnancy. Question 8: Why is there no absolute reference value for antibody screening? TORCH virus infection is a mother-fetus dynamic process and there is no definitive standard for each time period. Question 9: Why is quantitative analysis an advancement and the best choice for TORCH screening? 1. The production of IgG or IgM in the body is a rapidly changing process when initial infection or recurrent infection occurs during pregnancy, and can only be detected by quantitative analysis of concentration changes. 2.Quantitative analysis helps to detect false-positive or false-negative results. 3, For those pregnant women who have not done the basic immune status assessment before pregnancy, choosing two time points (T1, T2) to detect IgG or IgM concentration (C1, C2) and calculating the gradient of concentration change per unit time can effectively detect the specific immune response of the organism by viral attack, but there is no reference value yet, and the more common one is C2/C1>4 times. All these must be predicated on quantitative detection. II. Cytomegalovirus (CMV) screening Question 10: Why is CMV screening done during pregnancy? CMV is the most common cause of intrauterine infection, with a prevalence of 0,2%-2,2% in live births. Infection is a common cause of sensorineural hearing loss and mental retardation. Screening of pregnant women can help physicians understand the immune status of pregnant women, provide health education to IgG-negative pregnant women to prevent infection during pregnancy, and provide dynamic monitoring and observation to prevent the occurrence of initial infection. Viral DNA replication monitoring in IgG-positive pregnant women during early and late pregnancy prevents recurrent infections from causing congenital infections through the placenta and neonatal infections through the birth canal or breast milk. Infants can develop congenital CMV infection despite maternal immunity (IgG-positive). Both initial and recurrent infection in the mother can lead to symptomatic CMV infection in the fetus. Congenital CMV infection in the infants of some immune mothers results in central nervous system damage. Screening pregnant women can help physicians make a diagnosis of the type of infection in pregnant women so that they can choose an effective time for prenatal diagnosis of the fetus. Early detection and intervention can be achieved. Question 11: How to diagnose initial and recurrent CMV infection during pregnancy? After initial CMV infection in humans, the virus enters a dormant phase and remains latent in the body. This virus can be reactivated in the body and is called a recurrent (secondary) infection. In addition, re-infection can occur in immune individuals exposed to exogenous new strains of the virus. Thus, recurrent or reinfection is defined as intermittent virus secretion in the host immune state. This may be due to reactivation of endogenous virus or exposure of the host to exogenous new viral strains. Recurrent infection and reinfection cannot be distinguished by serologic testing, but only by viral isolation and detection by molecular biological methods. One third of healthy women who are CMVIgG positive in the United States will be reinfected with a new strain of CMV within 3 years. Serological methods to diagnose initial CMV infection during pregnancy: 1. IgM positive + rise in quantitative IgG test, conversion to positive after 15 days, seroconversion occurs = initial infection. 2. IgM positive + low affinity for IgG (≤16 weeks) = initial infection. If the pre-pregnancy immune status is unknown, the diagnosis of initial infection should be based on the detection of specific IgM antibodies. However, IgM antibodies can be detected in 10% of recurrent infections and in serum several months after the initial infection. Therefore, the population with positive IgM antibodies may include both initial infection before pregnancy and recurrent infection, as well as false positives due to long-term holding of positive IgM antibodies. IgG antibody affinity analysis can help to know if CMV infection occurred within 3 months, i.e., if the test result is high affinity, it can be concluded that the initial infection occurred before 3 months, often suggesting a previous infection; if the test result is low affinity, it is highly likely that the initial CMV infection occurred within 3 months, often suggesting an acute initial infection. Detection of viral DNA can help us detect viral replication, but cannot distinguish between recurrent infection or initial infection; only those with positive IgG antibodies can present with recurrent infection. Usually an affinity index <30% is highly suggestive of a recent initial infection (within 3 months). Therefore, the serologic diagnosis of initial infection during pregnancy is based mainly on the phenomenon of seroconversion (appearance of specific IgG antibodies in previously seronegative pregnant women) or detection of virus-specific IgM antibodies accompanied by low-affinity IgG antibodies. The presence of a significant increase in IgG antibody titers and high IgG affinity (with or without the presence of specific IgM antibodies) during pregnancy in a pregnant woman with pre-pregnancy serum detection of IgG antibodies and no IgM antibodies may be considered the presence of a recurrent infection. Diagnosis of non-first infection (recurrence and reinfection) in pregnancy: 1. IgM positive + IgG positive + high affinity (≤16 weeks) = increased likelihood of non-first infection. 2, IgG positive and IgM positive/negative + high affinity (≤16 weeks) + CMV detected in urine/secretion/blood (isolated virus or PCR) = non-first infection. 3. 4-fold rise in CMV-specific IgG = non-primary infection Question 12: What is congenital CMV infection? What are the consequences of congenital CMV infection? Congenital infection is due to vertical transmission of CMV through the placenta. Vertical transmission to the fetus is possible with initial or secondary infection in pregnant women. After initial infection, the probability of intrauterine vertical transmission during pregnancy is 30-40%, while after recurrent infection this probability is only 1%. However, a statistical analysis of fetal congenital defects published by the CDC in 2009 showed that the rate of recurrent CMV infection during pregnancy was 75% and that CMV infection caused the 1st highest number of congenital defects. Ten to 15% of congenitally infected infants are born with symptoms, including intrauterine growth retardation, microcephaly, hepatosplenomegaly, bruising, jaundice, chorioretinitis, thrombocytopenia, and anemia. Between 20-30% of these infants will die, primarily due to diffuse intravascular coagulation, abnormal liver function, or repeated bacterial infections. Most infants with congenital CMV infection (85-90%) will not show signs or symptoms at birth, but 5-15% of these infants will develop sequelae such as sensorineural hearing loss, psychomotor retardation, and visual impairment. Question 13: How is the prenatal diagnosis of fetal CMV infection made? The diagnosis of fetal CMV infection should be based on the culture of amniotic fluid samples and PCR testing. When a pregnant woman is diagnosed with initial CMV infection, amniocentesis should be performed after 7 weeks of maternal infection and after 20-21 weeks of gestation to collect amniotic fluid for real-time quantitative PCR to detect viral DNA load, because only after 5-7 weeks of fetal infection, after renal viral replication, can the amount of virus secreted into the amniotic fluid reach the limit of detection. According to many previous reports in the literature, the risk of false-negative results in prenatal diagnostic operations that are performed too close to the time of maternal infection cannot be ignored. There is no consensus on whether to take amniotic fluid virus testing in cases of recurrent infection (where the risk of fetal infection is low). However, according to the literature, some cases of recurrent infection have been reported to have serious sequelae. Therefore, we can consider prenatal diagnosis of fetal CMV infection by amniocentesis even in cases of recurrent infection. Q14: Why is the diagnosis of fetal infection by testing fetal blood for IgM antibodies or DNA not recommended? Not only because of the high risk of cordocentesis, but also because many CMV-infected fetuses develop specific IgM antibodies only in late gestation, which makes the sensitivity of cordocentesis testing low. At 20-21 weeks of gestation, fetal blood IgM sensitivity is 50-80% and fetal blood DNA sensitivity is 40%-90%, while amniotic fluid DNA specificity and accuracy can reach 100%. Question 15: Why is CMV-DNA testing in maternal blood not recommended for initial infection? Because the positive rate of CMV-DNA in the blood of pregnant women with initial infection is 33.3%, while the positive rate of CMV-DNA in the blood of IgG-positive healthy women is also 33.3%. Therefore, the use of CMV-DNA in the blood of pregnant women for the diagnosis of infection is unreliable. Question 16: What is the management of the fetus after the diagnosis of CMV infection? Since the prenatal diagnosis of CMV infection is limited to amniotic fluid testing (e.g., virus isolation and PCR), it is not possible to predict whether the fetus will be symptomatic at birth. Therefore, once fetal infection is diagnosed, pregnant women should undergo a series of ultrasound examinations every 2-4 weeks to detect signs of CMV infection, such as intrauterine growth retardation, ventricular dilatation, microcephaly, intracranial calcified foci, ascites or pleural effusion, fetal edema, hypo- or hyperhydramnios, and enhanced intestinal echogenicity, findings that may help predict the fetal prognosis. This systematic ultrasound should be performed at a qualified prenatal diagnostic center. Fetal high-resolution magnetic resonance imaging may be helpful in the evaluation of prognosis, especially if cranial abnormalities are detected on ultrasound. However, it remains to be determined whether magnetic resonance imaging provides us with valid information about fetal CMV infection. Several studies have been reported on the clinical significance of viral load in amniotic fluid as a prognostic indicator, showing that CMV-DNA load values in amniotic fluid samples are significantly higher in symptomatic fetuses than in asymptomatic fetuses. However, there was a large overlap of load values in both groups. Therefore, it remains to be further confirmed whether quantitative determination of CMVDNA in amniotic fluid can be used as a prognostic indicator of CMV infection. III. Rubella virus (RV) screening Question 17: Why is it desirable for women who are pregnant to receive antibody testing before pregnancy? Rubella, also known as German measles, is a childhood disease. It usually presents as a mild self-limiting disease in the absence of pregnancy. However, when pregnant the virus can have devastating effects on the developing fetus and is directly linked to unpredictable miscarriages and severe congenital malformations. Pre-pregnancy antibody testing can be performed to find out if the body is immune to RV, and if not (IgG negative), vaccination can be given and pregnancy can be delayed until antibodies are developed. Vaccination or naturally acquired immunity generally protects the fetus from intrauterine infection. Question 18: What is congenital rubella syndrome (CRS)? What are the most common congenital defects and delayed onset of symptoms? CRS represents a neonatal symptom of prenatal RV infection in a fetus with multi-organ system involvement. vertical infection of RV through the placenta is very dangerous to the developing fetus, leading to spontaneous abortion, fetal infection, stillbirth, and fetal growth retardation. Most children with CRS present with persistent neuromotor deficits and may later develop pneumonia, diabetes mellitus, thyroid dysfunction, and progressive holoprosencephaly. The most common congenital defects and later symptoms appear. Rubella virus infection at birth symptoms and late clinical presentation RV infection of the placenta spreads through the vascular system of the developing fetus, causing vascular cytopathy and local ischemia of developing organs. The rates of fetal infection and birth defects are related to the week of gestation at the time of maternal infection, as shown in Table 4. When maternal infection/exposure occurs in early gestation, the fetal infection rate is approximately 80%, decreasing to 25% in late to mid-gestation, and increasing again in later stages, from 35% infection at 27-30 weeks of gestation to almost 100% infection after 36 weeks of gestation. The rate of congenital defects is 90% before 11 weeks of gestation, 33% between 11 and 12 weeks of gestation, 11% between 13 and 14 weeks of gestation, 24% between 15 and 16 weeks of gestation, and 0 after 16 weeks of gestation. Therefore, the risk of congenital defects after maternal infection is limited to the first 16 weeks of gestation, and the risk of CRS due to infection after 20 weeks of gestation is minimal, and the only sequelae of infection in late pregnancy may be fetal growth retardation (FGR) The only sequelae of late pregnancy infection may be fetal growth retardation (FGR) . Maternal infections before and after fertilization also do not increase the risk of CRS. Therefore, counseling regarding fetal risk and management must be individualized. Vaccination or naturally acquired maternal immunity generally protects the fetus from intrauterine infection, however, maternal reinfection with CRS has been reported. Therefore, the possibility of CRS should be considered in fetuses or newborns with clinical signs of congenital infection. No cases of CRS have been reported in maternal reinfection after 12 weeks of gestation. Question 20: How is maternal RV infection diagnosed? Accurate diagnosis of primary RV in pregnancy is mandatory and requires serologic testing, as many cases are subclinical in presentation. The determination of RV-specific IgG and IgM by serological methods is an easy, sensitive and accurate way to diagnose the following: 1. 4-fold increase in RVIgG antibody titer in serum samples during acute and recovery periods. 2. Positive RV-specific IgM antibodies. A positive maternal blood IgM is accompanied by a serologic conversion indicator, i.e., the appearance of IgG conversion from negative to positive. Or pregnant women with IgM(+) and IgG antibodies with 4-fold increase in two consecutive sera (between 15 days and 1 month). 3. Positive RV culture (viral isolation culture of the patient's clinical sample). Serological tests are best performed within 7-15 days of the appearance of the rash and repeated once after 2-3 weeks. Question 21: Is it possible to diagnose fetal RV infection? There is no established and stable diagnostic method. There are a few reports of RV-specific PCR testing of CVS samples for the prenatal diagnosis of intrauterine RV. This report confirms the superiority of chorionic villus samples over amniotic fluid samples because chorionic villus can be obtained in early pregnancy at 10-12 weeks, whereas amniotic fluid needs to be obtained at 18-20 weeks of gestation and cord blood at 28 weeks of gestation, when detection of fetal infection is no longer very relevant. Ultrasound is extremely difficult to diagnose CRS, and biometry is helpful in diagnosing FGR, but is not a good tool for diagnosing CRS because the nature of the malformations caused by RV varies, and fetuses exhibiting growth retardation should be considered for congenital viral infections, including RV. Question 22: What if a pregnant woman shows signs or symptoms of rubella-like illness and has been recently exposed to RV? RV exposure in pregnant women must be individualized based on gestational age and immune status at the time of exposure. Confirmation of acute RV infection in pregnant women is often difficult, the clinical diagnosis is unreliable, a large number of cases are subclinical in presentation, and the clinical features are very similar to those of other diseases. Figure 3 shows guidelines for the management of exposed pregnant women or pregnant women with symptoms similar to rubella. If a pregnant woman has symptoms similar to rubella or has been recently exposed to RV, the gestational week and immune status should be determined. Diagnosis is difficult in pregnant women who are seen 5 weeks after exposure to RV or 4 weeks after the appearance of the rash. If IgG antibodies are negative, then the patient is sensitive to RV. Therefore, there is no evidence of recent infection. If IgG antibodies are positive, indicating previous infection, this is when antibody levels are low, suggesting a distant infection, but it is difficult to determine the timing of the infection and the risk of fetal infection. It is recommended to measure IgM or repeat IgG antibodies to see if there is a significant increase or decrease. Question 23: Can I get vaccinated while pregnant? Do I need to terminate my pregnancy if I accidentally receive the vaccine early in pregnancy or if I become pregnant immediately after vaccination? Contraindications to rubella vaccination include fever, immunodeficiency, neomycin allergy, and pregnancy. The rubella vaccine virus may infect the fetus through the placenta. However, no cases of CRS have been reported in the offspring of pregnant women who were accidentally vaccinated against rubella in early pregnancy. Therefore, termination of pregnancy is not recommended for such pregnant women. Based on the potential risk of the vaccine to the fetus, women are advised to wait 28 days after vaccination before becoming pregnant. Question 24: Why does screening for RVIgM antibodies only during pregnancy give false results? Because a positive non-acute infection can occur for two main reasons: 1. True positives: patients continue to express IgM for years, often with IgM levels remaining low and stable, often accompanied by IgG positivity. 2. false positives: the specificity of the assay is limited. the range of RVIgM antibody reference values is determined by the known negative and positive specimens. As shown in Figure 4, the area below the red line is the cross-over zone, a region that cannot be interpreted by the reference method, about 3-5% of which will have a false positive IgM problem, if we do not use quantitative methods we can not distinguish between true and false positives. The main causes of this situation are RF interference, cross-reactivity, polyclonal stimulation, etc. Question 25: What should I do if I have a positive IgM antibody during rubella screening in pregnancy? Positive IgM antibodies during rubella virus screening in pregnancy can be handled according to the process in Figure 5. Q26: Why is prevention the best strategy to avoid CRS? RV infection in pregnant women has devastating effects on the developing fetus, and the key to prevention is universal vaccination of all infants and immunization of high-risk groups of women screened before pregnancy. Diagnose cases of infection whenever possible. Avoid exposure to RV as much as possible in early and mid-pregnancy, including in IgG-positive pregnant women. for primary infections that occur before 16 weeks, pregnant women should be counseled about the risk of vertical transmission and offered advice on termination of pregnancy. However, there is no intrauterine treatment for an infected fetus. Therefore, prevention remains the best strategy to avoid CRS.