Direct Quantification of Human Cytomegalovirus Immediate-Early and Late mRNA Levels in Blood of Lung Transplant Recipients by Competitive Nucleic Acid Sequence-Based Amplification

Abstract

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10 4 copies per 100 l of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.

Cytomegalovirus (CMV) Inactivation in Breast Milk: Reassessment of Pasteurization and Freeze-Thawing

“…Therefore, the presence of viral pp67 late mRNA detected by NASBA in cell-free milk whey after freeze-thawing is an important molecular approach to confirm the results of quantitative virolactia by microculture. Furthermore, quantification of viral pp67 mRNA by NASBA (14,18) in naturally infected milk may provide a more suitable alternative that requires further analysis.…”

“…Viral late pp67 mRNA was detected in naturally infected milk whey using the NASBA technique (NucliSens CMV, Organon Teknika, Boxtel, The Netherlands) (14). Therefore, immediately after preparation of milk whey, 100 L were added to 900 L of NASBA lysis buffer [4.7 M guanidinium thiocyanate, 46 mM Tris, pH 6.4, 20 mM EDTA, 1.2% (wt/vol) Triton X-100], and the mixture was stored at Ϫ70°C until further processing (13).…”

Clinical Utility of Viral Load in Management of Cytomegalovirus Infection after Solid Organ Transplantation

“…Its low to modest sensitivity has limited its use and application in clinical practice. Development of a more sensitive assay, and preferably one with quantitative capability, is needed (172).…”

Clinical utility of CMV early and late transcript detection with NASBA in bronchoalveolar lavages

“…In addition, 3 samples tested positive by IE mRNA alone and 5 were positive by pp67 mRNA alone, while 2 samples were positive for both IE and pp67 mRNA but SV culture negative. Given previous reports that late transcripts may be more indicative of replicating CMV than early transcripts (Matthews and Boehme, 1988;Greijer et al, 2001b), we examined whether their presence was associated with a correspondingly higher fluorescent foci count in the CMV SV assay. The median fluorescent foci counts for each of the CMV SV positive groups were 10 (range, 1->1000; n = 74), 18 (6-232; n = 5), 445 (14->1000; n = 5) and 394 (2-394; n = 15), for IE and pp67 negative, IE positive, pp67 positive, and IE plus pp67 positive, respectively.…”

“…Assay of CMV mRNA by nucleic acid sequencebased amplification (NASBA) has been investigated for the detection of active viral gene expression and replication in blood (Aono et al, 1998;Blok et al, 1998b;Gerna et al, 1999;Greijer et al, 2002;Gerna et al, 2003;Witt et al, 2000;Oldenburg et al, 2000;Pellegrin et al, 2000). Since late transcripts such as pp67 mRNA reflect a complete replication cycle of CMV, they could be indicative of disease in the lung (Meyer-Konig et al, 1995;Greijer et al, 2001b). We therefore sought to examine whether NASBA detection of either early (IE) or late (pp67) transcripts in BAL specimens from transplant recipients is predictive of CMV pulmonary disease.…”