Persistent maternal viremia after varicella infection during pregnancy as a possible cause of false positive prenatal diagnosis of fetal infection on amniotic fluid
Dr V. Mirlesse, Service de Médecine Fœtale, Institut de Puériculture et de Périnatalogie, 26 Bd Brune, 75014, Paris, France.
Congenital varicella syndrome is a rare but potentially serious condition1 leading to congenital limb hypoplasia, skin scars and various other visceral damage, including damage of the central nervous system. These complications occur mainly when women present with varicella during the first 20 weeks of gestation but cases have been described up to 27 weeks.2 Prenatal diagnosis using ultrasound alone may not be sufficient to exclude fetal complications. Prenatal diagnosis of fetal contamination by detection of Varicella zoster virus with polymerase chain reaction on amniotic fluid has shown to be a useful tool, but few large series have been reported.3 Polymerase chain reaction diagnosis may allow more appropriate perinatal care. To our knowledge, no information about false positive or false negative results of this assay has ever been reported in the literature.
We present a case of maternal varicella during pregnancy leading to a false diagnosis of fetal infection by polymerase chain reaction on amniotic fluid caused by the persistence of viral particles in maternal peripheral blood. Following this case, we analysed a series of 99 women who presented with typical varicella skin rash during the first 25 weeks of gestation. We tested the persistence of viral particles in maternal blood prior to amniocentesis to minimise the risk of a false positive result.
The patient was a 32 year old, gravida 3, para 2 with no significant medical history. At 25 weeks, she presented with a typical varicella maculopapulovesicular rash complicated by varicella pneumonitis 10 days later. She developed an acute respiratory failure and severe hypoxemia and spent two weeks in an intensive care unit requiring artificial ventilation and intravenous treatment with antiviral drugs (acyclovir 10 mg/kg iv every 8 hours for 10 days).
After the complete resolution of the pneumonia and the skin rash, an amniocentesis was performed by her practitioner at 30 weeks to exclude fetal infection. The amniotic fluid was slightly blood stained. The position of the placenta was not recorded. Polymerase chain reaction assay was performed in three different rooms and according to the following technique: briefly, after red cell elimination by a selective lysis buffer, 1.5 mL of amniotic fluid was centrifuged and DNA was extracted from the pellet using a heat-detergent extraction buffer (10 mM NaOH, 0.5% Tween 20, 0.5% NP40) for 10 minutes in boiling water. After centrifugation at 16,000 ×g for 10 minutes, 10 μL of the supernatant was used to perform the polymerase chain reaction using primers located in the ORF17 of the Varicella zoster virus genome (nt 24774 to 24793 and nt 24923 to 24942). Carryover was avoided by the use of dUTP instead of dTTP and the addition of uracil-DNA-glycosylase (UDG) to each polymerase chain reaction mixture. Amplification was performed after a 2-minute incubation at 50°C allowing UDG to destroy any previously amplified product. The result was positive. Viral isolation on cell culture from the amniotic fluid was not performed.
The patient was then sent to our prenatal care unit to evaluate the fetal prognosis in relation to the supposed prenatal infection. The ultrasound survey did not show any anomaly. The patient was very anxious that some anomalies may have been missed by ultrasound alone. Magnetic resonance imaging analysis of the fetal brain and eyes was also normal.
After discussion and informed consent, a fetal blood sample was performed at 32 weeks to look for fetal biological anomalies. Simultaneously, a second amniocentesis was performed avoiding the placenta. The fluid was clear. The second polymerase chain reaction assay on the amniotic fluid tested negative. The fetal blood testing revealed normal blood cell count, no thrombocytopenia, no elevation of gamma glutamyl transferase, no elevation of alpha interferon and negative specific anti-Varicella zoster virus IgM.
The patient was encouraged to continue with her pregnancy and gave birth to a full-term healthy boy. Neonatal evaluation was normal. The child presented with a clinical varicella at the age of three and is now a healthy 7 year old boy.
The discordant results on two successive amniotic fluid samples led us to suspect a false positive result on the first sample. Maternal blood was tested for Varicella zoster virus with polymerase chain reaction immediately after the second amniocentesis and tested positive. We suspected that persistence of Varicella zoster virus particles in maternal blood lasted longer than previously described, and amniotic fluid contamination can result in a false positive diagnosis of fetal infection.
Following the index case described above, we decided to investigate the duration of positive polymerase chain reaction in maternal blood after varicella during pregnancy. Our aim was to limit the risk of contamination of amniotic fluid by maternal blood. We studied a prospective cohort of women who presented with typical varicella skin rash during the first 25 weeks of gestation.
From June 1994 to April 1999, women referred to our centre for counselling regarding the risk of congenital varicella syndrome were informed of the low frequency of severe fetal lesions. The benefits and limitations of a monthly ultrasound scan were discussed along with the diagnosis possibilities by polymerase chain reaction on amniotic fluid. Maternal infection had been diagnosed on typical skin rash and confirmed if necessary by serological assay. None of the patients had received varicella zoster specific immunoglobulins. We have no record on the use of acyclovir, but at that time, this drug was not allowed during pregnancy in France. Among the patients referred, 99 women asked for an amniocentesis to exclude fetal infection. Gestational age at the time of maternal infection is presented in Table 1. After information, prenatal counselling and patient's agreement, we performed polymerase chain reaction testing on maternal peripheral blood at least four weeks after clinical varicella and before amniocentesis.
Table 1. Gestational age of maternal skin rash in the series of 99 patients with varicella.
When polymerase chain reaction in maternal blood was positive, the blood was repeatedly tested every 10 days. When the maternal blood polymerase chain reaction became negative, the amniocentesis was performed. In 14 cases (14%), maternal blood tested positive between 4 and 10 weeks after the skin rash. No maternal blood was tested before four weeks after the skin rash and no positive result was detected over 10 weeks after the skin rash (Table 2). Among the 99 amniotic fluid samples tested for Varicella zoster virus by polymerase chain reaction in this series, fetal infection was found in five cases (5%).
Table 2. Varicella zoster virus polymerase chain reaction on maternal lymphocytes.
In those five cases where the amniotic fluid was infected, the gestational age of maternal skin rash was 7, 12, 14, 17 and 20 weeks of gestation, respectively. Two pregnancies were terminated at the parents' request and according to French law. One of the two fetuses had severe ultrasound anomalies including intrauterine growth retardation, hyperechogenicity of the bowel and lungs. The second one had no ultrasound anomalies at the time of termination but was found to be positive by postmortem polymerase chain reaction testing of fetal lung and brain. Three children were born alive and healthy. All three of them had prenatal magnetic resonance imaging showing no anomaly of the brain and eyes development. One of them developed shingles at three months of age. Long term follow up data are not available.
Prenatal diagnosis of Varicella zoster virus infection during pregnancy relies on ultrasound and polymerase chain reaction assay of amniotic fluid. Following our previous report,3 prenatal counselling in our unit includes a discussion about the limits and the risk of each methods. The diagnosis of fetal infection usually results in a closer monitoring of the infected fetuses (including prenatal magnetic resonance imaging of fetal brain and eyes) and to a more thorough information given to the parents on the possibility of dermatomal skin scars.
Most women feel reassured by a negative test on amniotic fluid as no false negative result or secondary transmission has yet been reported in this context. We do not usually consider pregnancy termination for women when the infected fetus has no ultrasound anomalies. However, knowing that in case of prenatal Varicella zoster virus infection, fetuses can develop secondary lesions4 and that no treatment is yet available, each parents' demand should be individually considered.
Viremia in varicella has been described as biphasic with a first peak in the days following contact and a second peak preceding the appearance of the skin rash.5 More recent studies examined the viral load by real time quantitative polymerase chain reaction assay after DNA extraction from peripheral blood mononuclear cells.6 Viral load has been found to remain positive for about a week after the onset of the rash, with a reduced number of viral copies after antiviral therapy.
In the European collaborative study on prenatal diagnosis, maternal cell contamination was found in 0.2% of amniocentesis samples.7 More recent work based on fluorescent in situ hybridisation reports a median maternal cell contamination of 2% in uncultured fluid.8 This contamination is increased with transplacental amniocentesis, multiple needle insertion and inexperienced operators.
Experience in prenatal diagnosis of Varicella zoster virus on amniotic fluid includes only small series and false positive results have never been mentioned in literature although the possibility of fluid contamination by maternal cells is well known. Contamination of the amniotic fluid leading to a suspected fetal infection has also been reported in a case of maternal Hepatitis C.9 Spontaneous clearance of amniotic fluid after viral infection has never been described. In fact, natural history of congenital rubella infection and a few longitudinal studies on the quantification of viral DNA of herpes virus in the amniotic fluid show a progressive increase in the viral load during pregnancy.10
Our findings suggest that, in the absence of any antiviral treatment, positive viral polymerase chain reaction in maternal blood can last much longer than one week after the rash. Therefore, the contamination of the amniotic fluid by maternal peripheral blood during the amniocentesis may lead to a false positive result of fetal infection.
Testing for Varicella zoster virus on cell culture was not performed in this series as it is considered much less sensitive. We cannot therefore exclude a possibility that a positive polymerase chain reaction test on maternal blood correlates with the persistence of a potentially active virus. Further studies based on viral expression are needed to clarify this point.
In conclusion we suggest that, when prenatal diagnosis of Varicella zoster virus infection on amniotic fluid is considered, maternal blood should be tested to exclude viremia before any invasive procedure is attempted.