Mother-to-child transmission of congenital cytomegalovirus infection and diagnosis
Congenital cytomegalovirus (cCMV) is the most common congenital viral infection in humans. It refers to children born to CMV-infected mothers with CMV infection confirmed within 2 to 3 weeks of birth and is caused by intrauterine infection. Each year in the United States, approximately 27,000 serologically negative pregnant women become primary infected with CMV, and 30,000 to 40,000 infants are infected each year. A recent Meta-analysis showed that the incidence of congenital CMV infection in underdeveloped countries was 1.2% (0.9%-1.3%), while in moderately developed countries the incidence was 0.39% (0.3%-0.5%). Only 10% of congenital CMV infections are symptomatic infections, such as microcephaly, cerebral edema, intracranial or intra-abdominal calcification, etc. The morbidity and mortality rate is 30% to 40%. The remaining 90% have no clinical symptoms at birth, but 10%-15% will have sequelae, including hearing impairment and mental retardation. 20%-30% of children with auditory deafness are caused by CMV infection.
I. Mother-to-child transmission of cytomegalovirus
(A) Maternal morbidity
The rate of CMV infection in the population is high, and the rate of CMV seropositivity in women of childbearing age is related to their socioeconomic status. 55% of women of childbearing age in developed countries have CMV, while 85% in economically underdeveloped countries. Half of the women in the UK are serologically positive for CMV at antenatal screening. In China, the infection rate of women of childbearing age is 60%. An epidemiological survey in Wuhan, Shenyang and Shanghai in China showed that the CMV infection rate of pregnant women reached 79% and 97%, and the active infection rate was 11,23%. Infection in pregnant mothers may be primary, secondary, or re-infection with a different strain of CMV. It is thought that pregnancy itself may increase a woman’s susceptibility to CMV or lead to activation of latent infection. Active maternal infection during pregnancy (including primary infection, exogenous reinfection or reactivation of endogenous latent infection) can lead to fetal and neonatal congenital infection. Maternal HIV infection, prematurity, and hospitalization in the NICU are all high-risk factors for congenital CMV infection.
(ii) Route of mother-to-child transmission
CMV virus can be transmitted intrauterine through placental infection, during delivery through contact with obstetric secretions and blood, and after birth through breast milk and saliva. Intrauterine transmission is mainly through the placenta. Animal experiments have shown that the placenta is first infected after the mother is infected, and a large number of WBCs accumulate in the CMV-infected cells of the intervillous space showing characteristic inclusion bodies. Immediately afterwards, the virus crosses the placental barrier to the fetus. Another route is that infected WBCs enter the fetal circulation through the umbilical vessels causing hematogenous virus transmission.
Deng Dongrui et al. showed that virus inoculation could lead to infection in 100% of pregnant rats and 94, 57% of the offspring. The infected virus was mainly located in the cytoplasm of endometrial stromal cells, vascular endothelial cells and leukocytes, and neutrophils could carry the virus to the endothelial cells of uterine capillaries, which further infected the trophoblast of vascular endothelial cells of fixed villi, leading to placental infection and degenerative changes of the villi. This may in turn be one of the causes of abnormal growth and development of the offspring. CMV can be excreted from the urinary tract or the cervix during the second trimester of pregnancy, so the infant can be infected by contact with the secretions of the birth canal or blood during delivery; since CMV exists in maternal saliva, breast milk and other secretions, postnatal infection is mainly transmitted to the newborn through close contact with the mother and through breast milk.
(C) Factors affecting mother-to-child transmission
The adverse effects of CMV on the offspring by vertical mother-to-child transmission depend on the type of virus, virulence, tissue affinity, the developmental stage of the fetus, and the immune status of the mother and child. Both the prevalence and severity of fetal CMV infection are related to the timing of maternal infection during pregnancy. The fetal infection rate of those infected in early and middle pregnancy is high and the risk is serious. The factors affecting mother-to-child transmission are as follows.
1. primaryinfection or recurrentinfection: CMV infection in pregnant women during pregnancy includes primary infection and recurrent infection. Primary infection refers to the initial infection. Recurrent infection refers to endogenous latent virus activation (reactivation), or reinfection (reinfection) with an exogenous strain of a different virus, or a larger number of the same strain of virus. Both primary and re-infection can cause congenital CMV infection, with primary infection being more likely to be transmitted to the fetus than re-infection.
In a Meat analysis, Kenneson et al. showed that the rate of mother-to-child transmission of primary infection in pregnant women was 32%, while the rate of transmission of recurrent infection was 1,4%. Primary infection occurs in 1-4% of pre-pregnancy serologically negative women during pregnancy, and the risk of transmission of the virus to the fetus is 30%-40%; 10%-30% of pre-pregnancy serologically positive pregnant women are reinfected during pregnancy, and the risk of transmission of the virus is 1%-3%. The risk of re-infection is 1.3%. The risk of urinary excretion of the primary infection is 10 times higher than that of re-infection, and the proportion of infants with symptoms and long-term sequelae is higher than that of re-infection. Less than 10% of mothers of children with severe sequelae had recurrent infections during pregnancy, and 0.2%-1.5% of newborns born to pregnant women with recurrent infections had sequelae.
As early as the 1980s, Stagno et al. showed that the teratogenic effect of congenital CMV infection (hearing and central nervous system damage) was mainly due to primary CMV infection during pregnancy, and now studies show that both primary and recurrent infections have teratogenic effects, and that recurrent infections can also lead to neurodevelopmental abnormalities and sequelae such as auditory deafness. The majority of congenital CMV infections are caused by re-infection, which accounts for 30-50% of congenital infections. Mothers with re-infection are immune to CMV, so the virulence is reduced in fetal infections, but fetal infections cannot be completely prevented. In a study of 64 infants with acquired congenital CMV infection due to maternal reinfection, none were born with symptoms, and only 8% developed long-term sequelae. When a pregnant woman has AIDS, she is prone to transmit CMV to her fetus even if she is re-infected.
2. Maternal viral load: Huang Qili et al. used quantitative PCR to detect maternal viral load, which can predict the possibility of mother-to-child transmission. When the maternal serum CMVDNA level is less than or equal to 1O, intrauterine infection can be excluded, and when the serum CMVDNA level is ≥l0, the intrauterine fetal infection rate is 100%. The sensitivity and specificity were considered to be 82, 3% and 100%, respectively. However, the study by Li Ping et al. differed somewhat from this, in their study no intrauterine infection occurred in pregnant women with CMVDNA copy number <10, and the intrauterine infection rates were 18, 75%, 28, 57%, 33, 33% and 60% at DNA copy number lO, l0, 1O and 10, respectively. The level of CMVDNA in the blood of pregnant women marks the strength of their viral infectivity. When the amount of CMVDNA in blood reaches 2, 62X10 copies/ml or more, the possibility of intrauterine infection should be considered.
3, the time of infection during pregnancy: the time of infection of the fetus varies greatly in the damage caused to the fetus. When the mother is infected with CMV in early pregnancy, because the fetus is in the embryonic development period, the CMV that invades the embryonic cells replicates in the cells, interfering with the normal division and development of the embryonic cells, so that the development of the embryo is impeded, chromosomal mutations, which can cause congenital malformations, miscarriage and stillbirth due to the impaired differentiation and development of its later tissues and organs. However, because of the immaturity of leukocyte development, there is no leukocyte aggregation, inflammatory exudation and other reactions in the infected cells; after mid-pregnancy, CMV can cause intrauterine growth retardation, central system damage, stillbirth and stillbirth; in late pregnancy, CMV infection mainly causes damage to fetal target organs, and there are many target organs attacked by CMV, but the most vulnerable ones are liver, lung and kidney. The most vulnerable organs are liver, lung and kidney. In the middle and late pregnancy, because the development of leukocytes in the fetus has matured, in addition to the degeneration and necrosis of the organs attacked by CMV, there are also infiltration of neutrophils, lymphocytes, plasma cells and monocytes; the infected cells are affected by the massive replication of CMV, forming giant cell-like changes and intracellular inclusion bodies formed by CMV replication.
In late pregnancy infection, the fetus was born with normal development, but the placenta had severe lesions. The three newborns were congenital CMV infections. The rate of intrauterine transmission of primary CMV infection in early pregnancy was 30,8% (4/13). Therefore, maternal CMV infection in early pregnancy poses a greater risk to the fetus than primary CMV infection before pregnancy.
CMV infection can suppress the body’s cellular immune function in several ways, and thus can escape the body’s immune attack and remain latent for a long time.
4, socioeconomic status and breastfeeding: family economic conditions can also have an impact on CMV infection in pregnant women. Stagno et al. found through a study of 16,218 pregnant women that 64,5% of pregnant women in the high-income group had negative serum CMV antibodies (a susceptible group), of which 1,6% had primary infection during pregnancy; 23,4% of pregnant women in the low-income group had negative serum CMV antibodies, and 3, 7 % had primary infection. Intrauterine transmission rates were similar in both groups (39% and 31%, respectively). Among congenital CMV infections, 25% were caused by primary infection in the low-income group and 63% in the high-income group. Therefore, the incidence of primary infection is higher in the high-income group due to the high proportion of susceptible individuals, and the impact on the fetus is greater. More attention should be paid to the occurrence of CMV infection in these groups after pregnancy.
Cytomegalovirus is usually excreted in breast milk, and cytomegalovirus seropositive women can transmit the virus to their infants through breastfeeding. In full-term infants, most postnatal CMV infections through breast milk are asymptomatic and have a low risk of neurological and deaf sequelae. Therefore, the benefits of breastfeeding for term infants far outweigh the minimal risk of transmitting the infection, meaning that the benefits outweigh the risks, and therefore term infants can continue to breastfeed.
However, great care should be taken with preterm infants with low birth mass because of their increased risk of cytomegalovirus disease. In a Brazilian study, preterm infants <34 weeks of gestational age and <1500 g of birth mass were born to mothers who were seropositive for CMV, but the newborns were confirmed free of CMV infection at birth. These newborns were breastfed after birth, resulting in 21 cases (22,1%) being infected, 1O of which excreted the virus within 60 d after birth and 11 of which started excreting after 60 d. Correlation factor analysis showed that in these infants, their infection was not associated with blood-borne transmission, birth mass and vaginal delivery, but with breastfeeding for the first 30 d after birth (OR=4,5, P=0,02) or breastfeeding for more than 30 d (OR=7,9, P<0,01).
Only one of the neonates was symptomatic of the infection. It is clear from the above study that early and prolonged breastfeeding of preterm infants whose mothers were CMV seropositive increased their risk of CMV infection. In another foreign study including 151 mothers and their 176 preterm infants (gestational age <37 weeks or birth mass <1500 g), cytomegalovirus of breast milk could be examined by serology, viral culture and PCR. PCR evaluation showed that in 73 of 76 (93%) of lactating women seropositive for CMV, the virus was activated in the breast milk and transmitted to the infant in 37% , 27 of the 73 mothers had reactivated human cytomegalovirus in their breast milk transmitted to 33 infants with a mean time to infection of 42 d.
Several studies conducted on several continents have confirmed the risk of CMV infection in low birth weight and very low low birth weight preterm infants who received breast milk from CMV seropositive mothers. The infectiousness of cytomegalovirus in breast milk is effectively removed by pasteurization; freezing (20oC) thawing reduces the infectiousness of cytomegalovirus in breast milk samples in vitro and reduces the risk of breast milk transmission of cMV, but does not seem to completely eradicate the risk, thus further studies are necessary to examine the relative effectiveness of freezing and thawing compared to pasteurization in preventing cytomegalovirus infection in high-risk infants. Further studies are needed to examine the relative effectiveness of freeze-thawing compared with pasteurization for the prevention of cytomegalovirus infection in infants at risk. Therefore, there is no unified understanding of whether to breastfeed a neonate with detoxification without intrauterine infection, and I believe that it is best not to feed CMV-containing breast milk to preterm and immunocompromised neonates, as they are more likely to develop postnatal infection and the virus is not easily cleared after infection.
II. Diagnosis of CMV infection
(A) Diagnosis of CMV infection in pregnant women
CMV infection in pregnant women generally has no obvious clinical symptoms, less than 5% have symptoms, very few manifest as mononucleosis syndrome. Those with symptoms may present with persistent fever, myalgia, and swollen lymph nodes. Therefore, it is difficult for pregnant women to realize that they are infected and have passed on to the fetus, so it is important to first determine whether a pregnant woman has CMV infection. Pregnant women should preferably undergo CMV serology before pregnancy to determine if they have ever been infected with CMV. If the specific antibodies are negative, it means that they have not been infected with CMV and they are susceptible. If the specific antibodies change from negative to positive after pregnancy, it will help the diagnosis of the recent primary infection.
However, many pregnant women have not been tested for CMV-specific antibodies prenatally, so when specific antibodies are present in the blood, further confirmation of a primary infection should be made, which can help determine the risk to the fetus.CMV, IgM can be used as an indicator of acute or recent infection and can be present at the time of primary infection, but is also common in exogenous reinfection or reactivation of endogenous latent infection.IgM can persist IgM can persist for up to 18 months and can still be detected 6 to 9 months after the acute phase of the primary infection, and its sensitivity in diagnosing primary infection is only 70% because the results may vary with different kits.
It can also have false positives in other infections such as B19 and EBV infection. The serology of primary infection in most pregnant women is characterized by CMV-IgM positivity and IgG negativity, or a change from negative to positive IgM and/or IgG, or a significant increase in potency 3 weeks apart. Most women who have been infected with CMV before pregnancy and are seropositive for CMV-IgG can still infect the fetus transplacenta if CMV latent in the body during pregnancy is activated or if they are reinfected with exogenous CMV, at which point the IgM is positive.
CMV-IgG affinity (IgGavidity) is currently the most reliable indicator for determining primary infection. The antibody affinity represents the ability of multivalent antibodies to bind multivalent antigens. Antibodies produced during primary infection have a much lower affinity for antigens than re-infection, and as the immune response matures, the affinity slowly increases, so low affinity represents acute or recent CMV infection. Measuring the affinity of maternal blood IgG antibodies at 16 to 18 weeks of gestation helps to identify all pregnant women who can cause fetal and neonatal infections with a sensitivity of 100%. However, the sensitivity decreases significantly after 20 weeks of gestation to 62,5%.121 Revello et al. suggest that a low to high IgG affinity index (AI) during the first 3 months of CMV infection indicates recent infection, e.g., AI ≤ 50% and IgM positivity suggests recent primary infection; AI > 65% may indicate previous infection, and at 18 weeks of gestation The negative predictive value is 100% at 18 weeks of gestation, but at 2l-23 weeks of gestation the negative predictive value is 90,9%.
Therefore, this test should be performed in the first trimester of pregnancy. Most obstetrics departments only test for CMV and IgM and ignore the IgG test. Therefore, it is recommended that both CMV-IgM and IgG antibodies should be measured in pregnant women to determine the primary infection and re-infection. If primary infection occurs in early pregnancy, due to the high possibility of fetal infection, in view of the current situation that each family in China has only one child, pregnant women can be advised to terminate the pregnancy to avoid the birth of congenitally infected children.
(B) Diagnosis of fetal CMV infection
1. Amniocentesis: Regardless of the route of infection, the renal tubular epithelium seems to be the main site of viral replication. The infected fetus excretes the virus into the amniotic cavity through urination, so amniotic fluid is a good way to detect fetal infection. Amniotic fluid CMV isolation and PCR for CMVDNA can effectively distinguish an infected fetus from an uninfected one. If a pregnant woman is suspected of having a recent infection, especially in the first trimester of pregnancy, amniocentesis should be performed for prenatal diagnosis. Amniocentesis for CMV culture and qualitative and quantitative PCR tests are best performed at 21 weeks of gestation or after 6 weeks of suspected infection. The sensitivity and specificity of viral isolation are high (100% specificity) and the sensitivity of PCR technique for CMV and DNA detection is 79,6%.
Virus isolation combined with PCR testing is probably the best method for prenatal diagnosis, and the sensitivity of the combination of the two can reach 80% to 100%. However, virus isolation in amniotic fluid and/or a positive PCR does not predict whether the fetus will be symptomatic at birth or whether sequelae will be left behind. Postnatal clinical symptoms are related to the amount of HCMV. guerra et al¨ found that amniotic fluid was negative for CMV quantification at 21-22 weeks of gestation. 100% of fetuses or neonates did not develop infection; CMVDNA quantification <10 copies/ml, 81% of fetuses or neonates did not develop infection, ≥10 copies/ml, 100% of fetuses or neonates developed congenital infection; CMVDNA < 10/ml, 92% of fetuses or newborns do not show clinical symptoms of infection, ≥10 copies/ml, 100% of fetuses or newborns show clinical symptoms of infection.
2.Umbilical cord blood testing: Cordocentesis can detect CMV-specific IgM antibodies and quantify viral load in cord blood. Because the specificity IgM antibody can be measured after 21 weeks of gestation, its sensitivity is low, only 20% to 75%¨, but its specificity is as high as 100%. It can not be used as the only method to exclude virus transmission, but it is still helpful to diagnose fetal congenital infection. Because of the low positive rate of cord blood virus culture, the sensitivity of antigenemia is 57,9%, the sensitivity of viremia is 55,5% and the specificity is 100%.
Recently, quantitative CMV testing has been reported for prenatal diagnosis. Revello and Gerna¨ extracted cord blood and measured CMV viral load for prenatal diagnosis and found that 19 out of 36 primary infected pregnant women caused intrauterine transmission, and the sensitivity of fluorescence I PCR method was 82,3%. Ultrasound revealed significantly higher viral load in the group with gross abnormalities and in the group with biochemical/blood abnormalities of cord blood than in the group with subclinical infection . The sensitivity of cord blood diagnosis is lower than that of amniotic fluid, and it is an invasive test that increases the risk of spontaneous abortion, so it is rarely used for CMV diagnosis in China.
3.Continuous ultrasound: it can monitor the general development of the fetus and can detect gross abnormalities, such as enlarged ventricles, growth retardation, abnormal echogenicity in the fetal abdomen. The absence of abnormal findings does not exclude functional damage, and there are reports that ultrasound can only detect 5% of CMV-infected children.
(C) Diagnosis of CMV infection after birth
1, serological testing: In the diagnosis of congenital CMV infection, serological tests are less sensitive and specific than viral tests, and therefore are not recommended. The detection of CMV-IgG in newborns is a marker for the entry of maternal antibodies through the placenta. Since IgM antibodies cannot enter the fetus through the placenta, a positive CMV-IgM antibody within 2-3 weeks after birth indicates congenital CMV infection in newborns. However, CMV, IgM antibodies may have false positive and false negative results. CMV infection produces IgM antibodies inconsistently between individuals, with approximately 27% of individuals failing to produce IgM antibodies.
It is suggested that failure to detect IgM antibodies does not completely rule out CMV infection. Although positive CMV and IgM in newborns suggests congenital infection. However, the diagnosis must be confirmed by viral isolation, and a negative CMV-IgM does not exclude congenital infection. In the study by Roubing Shan et al¨, only 24, positive newborns were positive for blood quantitative PCR, while in the study by Yijuan Li et al, of the 48 infants diagnosed with congenital CMV infection, 7 were symptomatic and 41 were asymptomatic, and none were positive for CMV, IgM. In those children with serologically positive sensorineural deafness, only a diagnosis of past infection can be made but it is not certain when the infection occurred (congenital or acquired). Unless a low affinity for IgG is found, this would indicate a recent infection.
IgG affinity (IgGavidity) is useful in diagnosing acute infections in adults, but no information is available on IgG affinity in neonates.
2, Rapid culture technique: This method can detect CMV at 24-48 h. After adding diagnostic samples to monolayers of cells, antibodies labeled with immunofluorescence or immunoperoxidase are added to detect early antigen¨ citation produced by CMV-infected cells.
3. Virus isolation and PcR technique: Specimens such as blood, urine and saliva are taken and inoculated into fibroblasts, and the virus is isolated after culture. It is the gold standard for the diagnosis of CMV virus infection. However, it has not been widely used clinically because of its cumbersome operation. PCR is widely used for the detection of CMV genome, and its sensitivity and specificity depend on the range of CMV genes of the probes used and the method used. Although nested PCR is more sensitive, it also increases the risk of false positives. This method is sensitive but does not distinguish between primary and secondary infection. It has been suggested that a positive plasma CMV, DNA represents active infection. Since some components of the urine may inhibit some enzymes in the PCR mixture and affect the assay results, internal quality control should be done to avoid false-negative results.
Driedbloodspot: Used to retrospectively diagnose congenital CMV infection. Since congenital CMV infection requires blood or urine for viral testing 2-3 weeks after birth, and many infants with suspected congenital CMV infection do not show symptoms until 2-3 weeks after birth or even longer, it is difficult to determine whether congenital CMV infection is present. Since the vast majority of newborns delivered in the hospital are screened for genetic and metabolic diseases with dried paper blood taken within 2 weeks of birth, the blood in these papers can be tested for CMV, DNA by PCR. It is possible to retrospectively diagnose congenital CMV infection many years after birth. Negative results cannot exclude congenital CMV infection due to the small amount of blood in the paper slides, only 50-100’Ll, which limits detection.
4, cMv antigenemia detection: the use of enzyme-labeled antibody method to detect CMV antigenemia, using the indirect enzyme-labeled antibody method of mouse monoclonal antibody CIO/C11 (C10/C11 method), as well as human monoclonal antibody C7 direct immunoperoxidase method (C7 a HRP method). That is, a method in which the CMVpp65 antigen is detected by applying immunostaining to the leukocytes and detecting it. Since CMV antigenemia coincides with the onset of CMV infection or can be diagnosed one step ahead, the diagnosis of CMV antigenemia will help in the early diagnosis of CMV infection or can be used to predict its onset. It is now being widely used to predict the onset and determine the efficacy of CMV infection in kidney, heart, lung, liver and bone marrow transplants, as well as in serious CMV infections such as CMV retinitis and interstitial pneumonia in HIV-infected patients.
In the case of positive antigenemia, the diagnosis of intrauterine CMV infectious disease can be fully established. However, its sensitivity is only 45,5%. This indicates that even a negative antigenemia does not exclude the possibility of intrauterine CMV infection.
In conclusion, this method is not yet competent when using antigenemia alone as a diagnostic test for intrauterine CMV infectious disease.
Li Yijuan et al¨ vice examined blood CMV-IgM, peripheral blood leukocyte CMV antigen and salivary PCRCMV-DNA in 98 high-risk neonates whose mothers were positive for blood CMV-IgM during pregnancy within 14 d after birth, and found that 48 cases diagnosed with congenital CMV infection The sensitivity of the antigen test and PCR test were 75% and 54%, respectively. cMV antigen test is early, sensitive and quantitative, PCR method can detect latent infection, and the combined application of the two can improve the positive diagnostic rate, CMV-IgM method has a low sensitivity for the early diagnosis of congenital CMV infection. sensitivity is low.
Ancora et al. used cranial ultrasonography to predict neurodevelopmental abnormalities in 57 children with congenital CMV infection, and followed up those with normal cranial ultrasound to 3 months and those with abnormalities to 6 months. 12 (21%) of the ultrasound abnormalities were found, mainly including periventricular and ventricular calcification, ventricular enlargement, bursal formation, and small brain damage. brain damage. Moreover, children with laboratory and clinical signs of CMV were more likely to have brain ultrasound abnormalities. Cranial CT does not detect more abnormalities than ultrasound. MRI missed 2 cases of calcification (2/11), but found more abnormalities than ultrasound in 6 cases, such as neurological migration disorder cerebral white matter dystrophy, delayed myelination, and one cyst missed by ultrasound.
These children were followed for more than 1 year, and of the l2 children with ultrasound abnormalities, l died in the neonatal period from large artery embolism, 1 had ventricular enlargement and cyst formation, and the remaining 10 had a good prognosis. Therefore, in symptomatic congenital CMV infection, ultrasonography can be used as a screening tool to estimate prognosis, is more economical and convenient than other imaging methods, and can be done at the bedside.