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Brief Reports Indian Pediatrics 2002; 39:561-564 |
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Human Cytomegalovirus Perinatal Infections in a Tertiary Care Setting |
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C.T. Shoby
Human cytomegalovirus (HCMV) was initially isolated from patients with congenital cytomegalic inclusion disease. HCMV is now recognized as an important pathogen in all age groups. Approximately 1% of all newborns are congenitally infected with HCMV, making HCMV the most common congenital infection(1). Though the role of HCMV in the causation of congenital infection has been studied over the last five decades, the magnitude of the problem in India has not been adequately investigated. Hence, a pilot study was done to estimate the rate of detection of HCMV in perinatal infections in a tertiary care setting. Subjects and Methods A group of 51 neonates within three weeks of life had been assigned as cases according to the clinical profile, of HCMV infection. These were unexplained IUGR, idiopathic jaundice (ruling out iso-immunization with ABO, Rh, G6PD deficiency, shape abnormalities, polycythemia, concealed hemorrhage), neonatal cholestasis (conjugated hyperbili-rubinemia), hematological abnormalities (petechiae, purpura, thrombocytopenia, unexplained anemia), neurological abnormalities, (microcephaly, intra-cranial calcification, hydrocephalus, unexplained seizures, unexplained lethargy), ophthalmic abnormalities (retinitis, optic atrophy) and non-immune hydrops (ascites) which were considered indicative of HCMV infection(2). A control group of fourty seven neonates were also selected, randomly from healthy term neonates who were admitted in the nursery for observation. This study focused on identifying HCMV as a cause of congenital infections and so infants with history of transfusion were excluded to rule out HCMV infection acquired after birth. The recruitment of all cases and controls was from neonates who were admitted to the nursery from April 1999 to October 2000. Most of our urine samples (i.e., 32 (63%) of the cases and 43 (92%) of controls) were taken within 3 days of birth. A polymerase chain reaction (PCR) was carried out on urine samples from both sick and healthy neonates. Only one sample was taken for urine, multiple samples were not taken because of financial constraints. In addition, plasma IgM and IgG avidity index testing of both groups of neonates and their mothers were carried out. DNA was extracted from urine by using a commercially available kit QIAamp® DNA Mini Kit (QIAGEN, Germany). Known positive and negative controls were used beside the multiple distilled water blanks. The positive control used was cell culture supernatant of HCMV (AD169) infected fibroblast cells while non-infected cell culture supernatant was used as the negative control. This set of outer and inner primers was checked against Herpes simplex virus 1 and 2, Varicella zoster virus, as well as human herpes virus 6 genotypes A and B and found to give amplification of only HCMV and lower limit of detection was found to be 0.01 PFU per reaction. The nested PCR was carried out in 50 µL volume reactions essentially according to previously published procedure(3). IgM Enzyme Immunoassay (EIA) was done by an automated system using IMx system (Abbott Laboratories, Illinois, USA). All the plasma samples giving HCMV IgM indices more than 0.500 were treated with the IMx rheumatoid factor neutralization reagent. The neutralized sample was assayed again by the same procedure. IgG antibody avidity test was done using an HCMV IgG test kit (NOVUM Diagnostica, Germany). Plasma samples were tested in singletons; with the same samples added to wells in a second plate for carrying out the urea wash procedure. HCMV IgG test with and without urea denaturation used in this study was carried out as described earlier(4,5). The avidity index (AI) was expressed as a percentage using the following formula:
An AI less than 30% indicates low avidity antibodies, mainly found in recent infection(5). AI above 50% indicates high avidity antibodies, which are associated with endogenous reactivation or reinfection or past infection. Certain samples could not be tested for antibody assays as they were hemolysed or had an icteric tinge. Results The clinical profile for the 51 cases included in the study were analyzed, unexplained IUGR was found to be the comonest feature among 44 (86.3%) subjects. Idiopathic jaundice was next common among 10 (19.6%) subjects. Hematological abnormalities were found in 2 (4%) cases and neurological abnormalities in 3 (6%) cases. Neonatal cholestasis, ophthalmic abnor-malities and non immune hydrops were not found. This study focused on identifying HCMV as a cause of congenital infections and so infants with history of transfusion were excluded to rule out HCMV infection acquired after birth. Six (11.8%) of the 51 cases gave a PCR signal for HCMV in urine, while all the 47 controls were negative. IgM was positive for the mothers of four (7.8%) of the 51 cases but only one (2.1%) of the 47 controls. One (3%) of the neonates from the control group (n = 33) was positive for IgM ELISA but none of the cases (n = 28) were positive. Among the symptomatic neonates who were positive by the HCMV PCR, two of their mothers were IgM ELISA positive. The clinical features of six HCMV PCR positive children and the children (HCMV PCR negative) of the two IgM positive mothers are given in Table I. All the samples tested were positive for HCMV IgG ELISA except one mother-neonate pair from the case group. None of the samples tested had low IgG avidity index. Discussion Active primary infection of the mother in pregnancy is the major cause of congenital infection in the neonate. It is important to differentiate primary from past infection. IgM estimation of the mother or neonate is a poor indicator of primary infection. Other indicators of HCMV infection are the low avidity IgG antibodies that are consistent with primary infection(5). IgM positivity with low avidity IgG indicates primary infection; in contrast, despite IgM positivity in the presence of high avidity IgG the inference on infection status should be that of reactivation of HCMV. The IgM assay is often associated with low sensitivity in certain situations. Table I- Clinical Features of Six HCMV PCR Positive Children and Children (HCMV PCR negative) of the Two IgM Positive Mothers
# Breech, fetal distress; * Artrial septal defect, small ventricular septal defect, patent ductus arteriosis and right sided pneumonia.
The previous experiences with plasma testing for IgM in our laboratory was good showing a concordance with nested PCR findings of HCMV infections in immuno-suppressed renal transplant patients(6). Hence, for convenience we tested plasma in this study from heparinised blood samples. Almost all the mothers tested in this study were positive for IgG ELISA test, which is consistent with earlier studies on adults from this region(7,8). A recent study from North India shows the IgM to HCMV was detected in 1.8% in cord blood from 1302 neonates(9). The frequency of detection of HCMV infection was 11.7% by nested PCR among congenitally sick neonates. IgM positivity in the mother was seen in four (66%) of these cases. IUGR was the principal clinical finding documented in 86.3% of infants in the study reported here. In this study IgM testing in neonates did not additionally pickup HCMV infection in this group. This study shows the value of molecular detection for HCMV to establish an association with symptomatic disease. It must be emphasized that HCMV testing on urine by PCR must be done early, preferably within seven days to definitively link the virus to congenital infection. In our group of six positive children, the urine was collected in all cases before day seven. IgM testing of neonates will have value only in the absence of blood or blood product administration. This was emphasized in a study carried out in our center(10). To comprehensively establish the HCMV association in suspected congenital infection, we recommend both nested PCR on urine and blood IgM testing of the baby taking any one parameter as definitive evidence. Contributors: CTS collected samples, performed assays and drafted the manuscript. RS, KAK and AKJ fomulated clinical criteria, selected the subjects and designed the study. MA and GJF designed the study and standardized the techniques. RK guided serology testing and co-drafted the manuscript. SCK assisted with performance of PCR. GS was responsible for overall guidance, interpretation of results and drafing of manuscript; he will act as the guarantor for the paper. Funding: Christian Medical College, Vellore, Fluid Research Fund. Competing interests: None stated.
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