significantly increased rate of lymphocytes !40% (39.2% vs 5.7% or 3.7%, respectively, P < 0.001) and elevated aspartate aminotransferase and/or alanine ...
BJOG: an International Journal of Obstetrics and Gynaecology June 2003, Vol. 110, pp. 572– 577
Clinical manifestations and abnormal laboratory findings in pregnant women with primary cytomegalovirus infection Giovanni Nigroa,b,*, Maurizio M. Anceschib, Ermelando V. Cosmib, The Congenital Cytomegalic Disease Collaborating Group The names of collaborators may be found on page 576 Objective To compare the clinical manifestations and laboratory abnormalities associated with primary cytomegalovirus (CMV) infection in pregnancy with recurrent and non-active CMV infection (controls). Design A prospective cohort study. Setting Rome, Latium and other Italian regions. Population Three hundred and sixteen pregnant women with CMV infection: 102 had primary infection, 105 had recurrent infection and 109 with non-active infection were followed up as controls. Methods CMV diagnosis was based on serological examinations (CMV IgG, IgM and IgG avidity) and detection of CMV DNA by polymerase chain reaction in maternal serum, urine and cervical samples. The clinical history and laboratory evaluations were carried out at enrolment and at each subsequent visit, every one to three months. Main outcome measures Identification of clinical and laboratory indicators of primary CMV infection in pregnancy. Results Compared with women with recurrent or non-active infection, women with primary infection had a statistically significant higher prevalence of fever, asthenia, myalgia and flu-like syndrome ( P < 0.001). In particular, relevant symptomatology was observed in 32 women (31.4%), of whom 25 had flu-like syndrome and 7 persistent fever as a single manifestation. Moreover, women with primary infection showed a significantly increased rate of lymphocytes 40% (39.2% vs 5.7% or 3.7%, respectively, P < 0.001) and elevated aspartate aminotransferase and/or alanine aminotransferase levels (35.3% vs 3.9% or 0.9%, respectively, P < 0.001): lymphocytosis and/or increased aminotransferases occurred in 53 patients (52%). In total, clinical manifestations and/or laboratory abnormalities occurred in 61 women with primary infection (59.8%) compared with 20 with recurrent infection (19%) and 13 controls (11.9%) ( P < 0.001). Conclusion Clinical manifestations (i.e. flu-like syndrome, fever) and abnormal laboratory findings (i.e. lymphocytes 40%, elevated aminotransferases) may suggest the presence of primary CMV infection and should prompt subsequent virological investigations. INTRODUCTION Cytomegalovirus (CMV) is the most common congenital infection and a leading cause of neonatal morbidity, including major long term sequelae such as mental retardation and sensorineural defects. In fact, of CMV-infected neonates who represent 1% of all live births, about 10% are symptomatic and another 10% will exhibit clinical manifestations later in life1 – 7. The nature of maternal infection is an important pathogenic determinant of congenital CMV
a
Paediatric Institute, La Sapienza University, Rome, Italy Department of Gynaecological Sciences, Perinatology and Child Health, La Sapienza University, Rome, Italy b
* Correspondence: Professor G. Nigro, Via dei Villini 35, 00161 Rome, Italy. D RCOG 2003 BJOG: an International Journal of Obstetrics and Gynaecology doi:10.1016/S1470-0328(03)01902-5
infection. Although a substantial proportion of women according to age, parity, ethnic and socio-economic factors are seropositive before pregnancy, a significant fraction become pregnant without any previous exposure to the virus8. Among susceptible women, the risk of seroconversion during pregnancy averages 0.7 – 4.1%8 – 11. In particular, the rate of CMV seroconversion in pregnant women from South European countries (such as Italy) and low income group women was estimated to be higher than that of childbearing women from North Europe and middle/high income group mothers from the United States12,13. A high prevalence of symptomatic congenital CMV infection may follow recurrent maternal infection as a result of elevated diffusion of different CMV strains in both young and adult people14,15. Recurrent maternal CMV infection has been recently reported to cause symptomatic congenital infection more frequently than previously believed7,15 – 17. However, primary infection is more likely to be transmitted to the fetus www.bjog-elsevier.com
CMV SYMPTOMATOLOGY IN PREGNANCY
than recurrent infection and may be responsible for severe, even lethal, fetal infection in about 8% of cases. In fact, about 20% of children show early or late manifestations due to maternal – fetal CMV transmission, which occurs in about 40% of women with primary infection6,7,10,13. The differentiation between primary and recurrent infections is therefore of importance for the management of pregnancy. The most common clinical manifestation of primary CMV infection in adults is a heterophilic, antibody-negative mononucleosis-like illness that is reported to occur in about 10% of the patients5,9,18,19. Given the paucity of data regarding the occurrence of clinical manifestations associated with CMV infection in pregnancy, particularly recurrent infection, we have studied a cohort of patients with primary or recurrent CMV infection and compared them to women with non-active infection.
METHODS Between April 1994 and March 2002 a total of 316 pregnant women were referred to our tertiary centre with possible active CMV infection. Usually, this was because of positive or doubtful CMV IgM antibodies or, less frequently, high IgG antibody levels. In fact, serological screening for CMV in pregnancy, or even before, is increasingly requested by the gynaecologists in Rome and most of Italy. Few women were referred after ultrasonographic evidence of possible fetal involvement or virological demonstration of fetal CMV infection. When first seen, a complete history was taken and particular care was given to the type and timing of symptoms the patient had experienced during pregnancy (i.e. if they had had episodes of possible illnesses, including fever, rhino-pharyngo-tracheitis and flu-like syndrome). Fever was considered as body temperature 37.5jC for at least two days or 38.5jC if lasting only one day. A flu-like syndrome was defined as the simultaneous occurrence of fever and rhinitis and/or pharyngitis and/or tracheo-bronchitis with asthenia and/or myalgia. The clinical manifestations and laboratory data that were subsequently analysed for prevalence and significance were the ones as close as possible to seroconversion or recurrence (more or less 2 weeks). For controls, all the manifestations occurring from the beginning of pregnancy until enrolment were considered. Blood samples were withdrawn for serological (CMV IgG, IgM and IgG avidity) and virological examinations (CMV DNA detection by polymerase chain reaction). Urine and cervical samples (taken with a sterile swab) were also examined for CMV DNA detection. The patients were then followed by monthly clinical, laboratory and ultrasound examinations concomitantly with virological and serological investigations for CMV. DNA was extracted from clinical samples as described previously16. The CMV DNA sequences of exon 4 of the IEA1 gene located in the EcoRI J fragment of strain Ad169 D RCOG 2003 Br J Obstet Gynaecol 110, pp. 572 – 577
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were amplified from amniotic fluid and the other biological samples14. The polymerase chain reaction was performed as a nested polymerase chain reaction with two sets of primers. The outer primers set consisted of C nucleotides 1729– 1748 complementary to the antisense strand and D nucleotides 1951 – 1970 complementary to the sense strand. The nested primer set consisted of A nucleotides 1767– 1786 complementary to the antisense strand and B nucleotides 1893– 1912 complementary to the sense strand. The outer primer set C –D amplified a 242 bp amplimer and the nested primer set A – B amplified a 146 bp amplimer. Negative controls including reagent blanks and CMV-seronegative sera were examined every two or three test samples for all reactions. The DNA extracted from purified CMV strain AD 169 was used as positive control at three different concentrations. The specificity of the primers was verified on DNA of non-infected human sera, urine, placenta and other herpes viruses (herpes simplex virus [HSV], varicella-zoster virus [VZV], Epstein – Barr virus [EBV] and human herpes virus type 6). To avoid contamination of the polymerase chain reaction mixture, all reactions were performed under stringent precautions, including the use of fitted latex gloves, sterile pipette tips, sterile reagents divided into aliquots and a separate set of pipettes in a separate room for the pre-polymerase chain reaction handling of specimens. To prevent polymerase chain reaction product contamination, each microtube was spun in a microcentrifuge for 5 seconds before opening the cap. CMV-specific total immunoglobulin (IgG, IgA and IgM) antibodies were detected by CMV total Ig Abbott (North Chicago, Illinois); class-specific CMV antibodies (IgG and IgM) and CMV IgG avidity were measured by enzyme immunoassays from Radim (Pomezia, Italy), according to manufacturer’s instructions. The classification of the patients into the three diagnostic groups was made at completion of laboratory and virological examination according to the following criteria16. Primary infection was diagnosed by seroconversion, as shown by the occurrence of increasing levels of CMV IgM and/or IgG antibodies and low IgG avidity in previously CMV-seronegative subjects. Recurrent (reactivation or reinfection) infection was demonstrated by a significant increase in CMV IgG titre (at least double that tested previously) associated with high IgG avidity and positive IgM antibodies or CMV DNA detection in serum, urine or cervical samples in women who were CMV-seropositive before pregnancy. In patients with positive CMV IgM antibodies, possible interference by rheumatoid factor or other herpes viruses (HSV types 1 and 2, EBV, VZV) was excluded. Non-active CMV infection was shown by CMV seropositivity without significant antibody changes or CMV IgM or DNA detection in pregnant women who were already seropositive before pregnancy. Based on the previous criteria, 102 patients were diagnosed as having primary infection and 105 had recurrent infection. The
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Table 1. Prevalence of clinical manifestations in pregnant women with primary or recurrent CMV infection and in controls. Values are given as n (%). Clinical manifestations Fever Asthenia Myalgia Rhino-pharyngo-tracheo-bronchitis Flu-like syndrome
Primary infection (n ¼ 102) 43 32 22 43 25
Recurrent infection (n ¼ 105)
(42.1) (31.4) (21.5) (42.1) (24.5)
18 12 7 31 10
Controls (n ¼ 109)
(17.1) (11.4) (6.7) (29.5) (9.5)
14 9 6 33 7
(12.8) (8.2) (5.5) (30.3) (6.4)
m2
P
27.367 23.386 16.910 4.842 6.895
40 iu/L)
Primary infection (n ¼ 102)
Recurrent infection (n ¼ 105)
Controls (n ¼ 109)
P
7.9 [6.2 – 9.0] 34.5 [29.0 – 43.0] 40 (39.2) 6.0 [4.0 – 7.0] 199 [169 – 231] 22 [16 – 38]b 24.5 [15 – 61]c 36 (35.3)
8.0 [6.57 – 9.4] 25.0 [21.0 – 30.0] 6 (5.7) 5.0 [4.0 – 6.0] 219 [195 – 251] 16 [14 – 18] 15 [12 – 19] 4 (3.9)
8.1 [7.0 – 9.9] 24.0 [20.0 – 29.0] 5 (3.7) 5.0 [4.0 – 6.5] 225 [189 – 257] 16 [13 – 20] 15 [11 – 20] 0 (0)
0.165*
