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September2004 Vol.41 Issue:        3       (Supp.) Table of Contents
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Quantification of Cytomegalovirus DNA by PCR: Clinical Applications

Ahmed A. Dardir1, Lamia El-Houseny2, Mostafa K. El Awady2

 

Departments of Research on Children with Special Needs1, Biomedical Technology2,

National Research Center, Cairo



ABSTRACT

Cytomegalovirus (CMV) is the most common vertically transmitted viral infection in the developed world. Congenital CMV infection occurs in 0.5–2.0% of all deliveries and remains a major problem all the life long. Molecular biology techniques have enriched knowledge of CMV infection. Assays for CMV antibodies are of little clinical value as they lack specificity for the diagnosis of viremia and the results obtained do not always correlate with clinical symptoms.The aim of this study was to evaluate quantification techniques for CMV load in peripheral blood as a surrogate marker for disease severity and treatment follow up. We selected 20 infants diagnosed as congenitally infected with CMV and 50 mothers who tested positive for CMV IgM and/or high IgG titre and positive PCR for CMV-DNA, to be included in the present study. Neonatal manifestations were recorded in 85% of the affected infants. Developmental delay (90%), mental subnormality (75%), microcephaly (45%), seizures (40%) and sesorineural hearing loss (25%) were the most frequent clinical findings. Cases with chorioretinitis (20%) were associated with the most devastating cognitive deficits and severe microcephaly. Brain neuroimaging showed that cortical and central atrophic changes were the most predominant findings (60% of cases) followed by white matter hypomyelination (35%), intracerebral calcifications (20%) and cerebellar hypoplasia (10%). However, normal brain neuroimaging (15% of cases) did not exclude CMV infection. Frequent presenting complaints of mothers included delivery of an affected infant (40%), repeated abortions (30%), repeated neonatal deaths (20%) and repeated intra-uterine fetal deaths (10%).Viral end-point dilution using nested PCR for CMV-gpB-DNA was done for all mothers and infants. Oral ganciclovir was administered to 12 mothers at a dose of 1000mg tid for 14 days and CMV loads were followed up by end-point dilution nested PCR before and after treatment. Our results indicated that viral load below the level of 1.6 Х103 GE/ml is not accompanied by symptomatic CMV disease. High viral load was associated with severe clinical picture of the affected infants; however, such an association was not present in the mothers. Moreover, CMV IgM was negative in cases with very low viral load denoting that end-point dilution nested PCR is a more sensitive method in cases with very low CMV load. Five mothers out of 12 who received ganciclovir treatment did not respond to therapy whereas 2 of them had high viral load and delivered severely affected infants with congenital CMV denoting that increasing levels of systemic CMV load in pregnant mothers precede onset of  severe CMV disease in their newborns. Real-time PCR of CMV-UL83 gene was done for 20 cases (17 mothers and 3 infants). Detectable viremia was found only in 5 patients (3 mothers and 2 infants) with poor correlation with corresponding results of end-point dilution method for the same patients. Since UL83 gene levels are known to correlate with replicating viremia, we conclude that 25% of these 20 cases had replicating viremia and are therefore, mandatory for treatment.  In conclusion: End-point dilution nested PCR is a sensitive method in detecting very low CMV quantity which is helpful in accurate diagnosis and management. The amount of CMV DNA in peripheral blood is considered a direct marker for CMV infection and elevated viral load is the predominant risk factor for progression to CMV disease. Real-time PCR for UL83 gene is another important method for detection of CMV activity. So that, both viral load and viral activity are important markers in differentiation between latent and active infection.

(Egypt J. Neurol. Psychiat. Neurosurg., 2004, 41 (3) suppl.: 853-867).

 





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