Detection of Ganciclovir Resistance in Patients with AIDS and Cytomegalovirus Retinitis: Correlation of Genotypic Methods with Viral Phenotype and Clinical Outcome

Abstract

Because blood specimens directly amplified by PCR can be analyzed more rapidly than can cultures (< or =48 h vs. > or =4 weeks), sequencing the CMV UL97 gene from blood specimens directly amplified by PCR may be useful clinically.

Rapid Detection Directly from Patient Serum Samples of Human Cytomegalovirus UL97 Mutations Conferring Ganciclovir Resistance

“…These primers amplify a 921-bp region of UL97 (from codons 400 to 707), and we reasoned that the efficiency of amplification by these primers might be limited by their length. Our hypothesis was supported by the results of Spector and coworkers, who used seven overlapping primer sets to amplify and sequence the relevant region of UL97 directly from plasma from 10 patients with known ganciclovir-resistant CMV infections (10,11), and by the results of Jabs and coworkers, who used a nested PCR approach to amplify this region for sequencing directly from patient materials (6,7). Since the use of seven primers sets would be impractical for most clinical laboratories and to avoid the need for nested PCR, we designed two optimized primer sets allowing amplification of this region: forward primer 5Ј-TGG CCG ACG CTA TCA AAT TT-3Ј and reverse primer 5Ј-CCC AGC GCC GAC AGC TCC GAC-3Ј, amplifying codons 439 to 557, and forward primer 5Ј-ATG TCG GAG CTG TCG GCG-3Ј and reverse primer 5Ј-CGA CAC GAG GAC ATC TTG-3Ј, amplifying codons 550 to 645.…”

“…In one specimen, the new method identified one mutation (D605E) that was not detected by the traditional assay. In a recent study evaluating this issue, there was Ͼ90% agreement between UL97 sequences directly amplified from clinical specimens and those from culture isolates (7). In the case of discrepancies, it is likely that the results of an assay performed directly on patient specimens more closely reflect the clinically relevant viral species.…”

Change over Time in Incidence of Ganciclovir Resistance in Patients with Cytomegalovirus Retinitis

“…Culture isolates, rather than directly PCR-amplified blood specimens, were used to detect resistant CMV, because culture results are more predictive of clinical behavior [22]. For culture isolates, measures of phenotypic and genotypic resistance are highly correlated [23] and are similarly predictive of clinical behavior [24].…”

Recent developments in human cytomegalovirus diagnosis

“…Although selective PCR-sequencing of UL97 and UL54 provides rapid and comprehensive results, genotypic assays can miss some resistances detected by phenotypic assays [69,71]. Conversely, the presence of mixed viral populations in blood samples and the potential selection bias introduced by in vitro susceptibility testing may underestimate the real impact of HCMV resistance in patients failing antiviral therapy [70,86]. Therefore, a combination of phenotypic and genotypic assays is recommended for complete analysis of HCMV antiviral resistance [70].…”