Comparison of in situ hybridization and monoclonal antibodies for early detection of cytomegalovirus in cell culture

Abstract

The abilities of each of four diagnostic tests-direct fluorescent monoclonal antibody (direct FA) staining, indirect fluorescent monoclonal antibody (indirect FA) staining, in situ hybridization with biotinylated DNA probes, and in situ hybridization with DNA probes directly linked to enzymatically active horseradish peroxidase-to detect cytomegalovirus soon after culture were compared. Only the indirect FA test and the in situ hybridization method with directly linked HRP-DNA probes provided consistent and reliable cytomegalovirus detection as early as 15 h postinfection.

Optimization of in situ hybridization for detection of viral genomes in cultured cells on 96-microwell plates: a cytomegalovirus model

“…In our investigations, we found that in cells infected with clinical samples, ISH failed to detect viral sequences during the first 24 h after inoculation but gave a signal of progressive intensity in the following 36 or 48 h and was successful in detecting viral genomes at 72 h p.i., confirming the results obtained by Sorbello et al (30). These observations permit us to eliminate the possibility of methodological artifact (17).…”

“…The high efficiency of alkaline phosphatase-conjugated avidin as a detection system is not surprising, since this enzyme has been repeatedly proven to be very sensitive for immunohistochemical detection of mRNAs (9, 29). The lower sensitivity of the results obtained by McClintock et al (17), Gleaves et al (7,8), and Scott et al (28), who used the same Enzo probe, may result from the use of an alternative method for signal detection, a chromogenic substrate solution containing aminoethylcarbazole-H202-acetate.…”

“…Since the diagnostic value of serologic responses to CMV is controversial (18,23,24,26,35), virus isolation appears to be the most reliable indicator of CMV infection (22,23,25,26,35). The time-consuming tissue culture method for detection of viral cytopathic effects has been replaced by alternative methods, which include detection of antigens by immunofluorescence (6,17,18,21,27,30,32,(35)(36)(37) or by peroxidase staining (16,22,34,35). Recently, authors have been able to detect nucleic acid sequences of CMV directly from clinical specimens (8,14) or from peripheral blood leukocytes (5) by in situ hybridization with radiolabeled (5) or biotinylated (8,14) probes to simplify and accelerate the diagnosis of CMV infection.…”

“…However, these techniques do not confirm the actual infectivity of the virus. Previous studies have shown that detection of nucleic acid synthesis of CMV in shell vial-cultured samples (7, 17,28) and DNA hybridization are as specific and nearly as sensitive as culture but not sufficiently rapid to replace the use of monoclonal antibodies (MAbs) (7,17,28).…”