In our patient population, a commonly used CMV Q-PCR that targets the gB gene (14
) failed to quantify the virus in 11% (6/54) of isolates. This was most likely due to a known variant, C630T, in four isolates, which allowed qualitative detection of the virus (15
). However, in two isolates sequence variation in the target sequence of the probes made the viral genome invisible to the assay and created a true false-negative result.
The frequency of the gB C630T variant in our small survey was similar (7.4% versus 6.1%) to the frequency reported by Schaade et al. (15
). In a population of primarily bone marrow transplant recipients (85%) in Portland, Oregon, the frequency of the gB C630T variant was reported to be 2.3% of patients (8
). In a study of 66 solid organ transplant recipients, no C630T variants were found (12
). Although the gB Q-PCR assay is unable to quantify CMV with the C630T variant, it still detects the virus.
Far more serious is our finding of two CMV isolates that are more divergent than the C630T variant. These isolates have several additional sequence differences with the probes used in the gB assay (Fig. ). Due to the additional sequence differences, these CMV isolates are undetectable by this assay.
Additional sequence analysis of the two CMV isolates that produced a false-negative result in the gB Q-PCR (Fig. ) revealed that both isolates are not new but are most likely the same as the previously discovered gB genotype 5 (gB5) (16
). This genotype was defined by sequence analysis of two variable regions in the CMV glycoprotein B gene and was found to be distinct from the other four gB genotypes of CMV (4
). The two isolates derived from our patient population are identical or nearly identical to the upstream variable region of the gB5 consensus sequence (Fig. ) (16
). We did not study the downstream variable region of the CMV gB gene in our isolates. The additional differences between the two isolates studied here (GenBank accession numbers DQ121372
) and other published CMV sequences (Fig. ) reveal that gB5 is likely to be more divergent than previously thought. In particular, the sequence divergence extends outside of the previously known gB variable regions (4
). The sequence diversity that we found among our clinical isolates adds to our knowledge of sequence diversity in the CMV gB gene and consequently should be taken into consideration in the design of Q-PCR assays that use this gene as a target.
CMV with the gB5 genotype has not been reported frequently. After its original discovery (16
) in five patients with AIDS, another group (3
) reported sequence data consistent with a gB5 genotype in five patients with AIDS. This may be due to the actual rarity of the strain and its confinement to AIDS patients, but the apparent rarity of gB5 may also be because it is difficult to detect. The primers used for genotyping by several groups are not able to detect gB5 because of its sequence divergence (4
In a comparison of sequence diversity among clinical isolates, the CMV DNA polymerase (pol
) gene was found to be slightly more conserved than the gene encoding gB, suggesting pol
may be a better target, in terms of clinical sensitivity, for determining CMV viral load (17
). We found that an adaptation for the LightCycler of the Q-PCR assay described by Yun et al. (18
), which targets pol
, successfully detected and quantified all 54 of our clinical isolates.
Real-time Q-PCR assays that use both primers and probes are inherently more vulnerable to false-negative results arising from sequence diversity, because they depend on more sequence than a simple PCR to achieve analytical sensitivity and specificity. Based on this principle and our experience, we suggest that laboratories carefully evaluate the clinical sensitivity of a candidate Q-PCR assay in their patient population to avoid errors due to sequence diversity in the target sequences. Consideration should also be given to using more than one target to avoid false negatives due to rare or newly arising variants that escape detection during the initial validation. This approach has been taken by several laboratories (7