3rd Conference on Retroviruses and Opportunistic Infections Washington, DC - January 28-February 1, 1996

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:55 (abstract no. 11)

Bolvin G, Handfield J, Murray G, Toma E, Lazar J, Bergeron M
Laval University, Quebec City, Qc, Canada.

About 90% of patients with AIDS will develop active CMV infection and up to 40% may experience life- or sight-threatening diseases due to this virus. We developed a Q-PCR assay to determine the CMV viral load in PMNL of HIV-infected patients (CD4 less than 200 x 10⁶/L) without CMV disease (n=53; group A) and with CMV disease (n=7) at the beginning (group B) and after 1 month of systemic anti-CMV therapy (group C). DNA from 1 x 10⁵ PMNL was amplified by PCR using biotinylated primers flanking a 294-bp fragment in the CMV Major Immediate-Early (MIE) gene. The Sharp Signal System (Digene Diagnostics, Inc.) was used for detection of the PCR products. This system consists of a non-isotopic detection assay using a specific CMV RNA probe and an enzyme-labeled antibody directed against RNA: DNA hybrids. Standard curves of the assay obtained after different substrate incubation times (30 mn, 1 h, 2h, 24h) were generated by plotting on a graph the optical density values against thenumber of CMV copies from a plasmid containing known amounts (25 to 50000 copies) of the CMV MIE gene. Over that range of copy number, the intra- and interassay variability was respectively 15% and 24% Mean number of CMV copies (range) per 1 x 10⁵ PMNL from patients in groups A, B, and C, was 366 (0-7160); 33894 (1120-119350); and <25 (O-68). Also, two-third of group A patients had negative PCR results (lower limit of detection: 25 copies).

CONCLUSION: Periodic assessment of CMV DNA load in PMNL by Q-PCR could be useful for identification of patients at high risk of developing CMV disease and for monitoring of the effects of antiviral therapy.

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