Rapid Genotyping of Cytomegalovirus in Dried Blood Spots by Multiplex Real-Time PCR Assays Targeting the Envelope Glycoprotein gB and gH Genes

Abstract

Genotyping of cytomegalovirus (CMV) is useful to examine potential differences in the pathogenicity of strains and to demonstrate coinfection with multiple strains involved in CMV disease in adults and congenitally infected newborns. Studies on genotyping of CMV in dried blood spots (DBS) are rare and have been hampered by the small amount of dried blood available. In this study, two multiplex real-time PCR assays for rapid gB and gH genotyping of CMV in DBS were developed. Validation of the assays with 39 CMV-positive plasma samples of transplant recipients and 21 urine specimens of congenitally infected newborns was successful in genotyping 100% of the samples, with gB1 and gB3 being the most prevalent genotypes. Multiple gB and gH genotypes were detected in 36% and 33% of the plasma samples, respectively. One urine sample from a newborn with symptomatic congenital CMV was positive for gB1 and gB2. DBS of congenitally infected newborns (n ‫؍‬ 41) were tested using 9 l of dried blood, and genotypes were detected in 81% (gB) and 73% (gH) of the samples, with gB3 being the most prevalent genotype. No clear association of specific genotypes with clinical outcome was observed. In conclusion, the CMV gB and gH PCR assays were found to be rapid, sensitive for detecting mixed infections, and suitable for direct usage on DBS. These assays are efficient tools for genotyping of CMV in DBS of congenitally infected newborns. C ytomegalovirus (CMV) is the most common cause of congenital infection worldwide and an important viral pathogen affecting immunocompromised patients (13, 21). Both in congenitally infected newborns and in immunocompromised patients, genotyping of CMV has been used to study potential differences in pathogenicity of specific strains. However, few authors describe a correlation between specific CMV genotypes and severity of disease (29,32,34,40). More important, genotyping of CMV has enabled the discrimination of reactivation of latent virus from reinfection with new CMV strains in transplant patients, allowing a better definition of donor-to-recipient transmission patterns (24). Congenital CMV infections mainly result from recurrent infections among pregnant women (37), comprising both reactivation and reinfection. The discrimination of reactivation from reinfection may give insight into the mother-to-fetus transmission pattern and the possible associations with the outcome of congenital CMV infections.Genotyping of CMV has mainly focused on envelope glycoproteins gB (UL55) and gH (UL75), which play a role in virus entry and are major targets for neutralizing antibody response. The most frequently used methods for genotyping of CMV are nucleotide sequence analysis (27) and restriction fragment length polymorphism of PCR products (25, 28). Recently, real-time PCR-based assays have been used for rapid detection and quantification of CMV gB and gH genotypes (12,15,24,26). However, they have mainly been applied to plasma or other high-volume samples. Also, deep-sequencing-based methods, sensitive in the de...

Diagnosis, gB genotype distribution and viral load of symptomatic congenitally infected CMV patients in Cuba

“…Furthermore, despite genotypic variability of CMV seems to be geographically related, different genotypification methodologies have proven different sensitivities for mixed infection detection. 26,27 Mixed infection with multiple CMV strains occurs in various patient populations, including immunocompetent and immunocompromised subjects. However, infection with multiple strains has rarely been described in NB with congenital CMV.…”

Cytomegalovirus Glycoprotein B Genotypes in Mexican Children and Women

“…So far, studies directed towards assessing the impact of genotype features on disease have yielded conflicting results. Nevertheless, it appears that in certain circumstances strain type may have a role in the occurrence of CMV disease; for instance, most studies in HIV-positive patients report gB2 as the most common genotype [4,6,15,16,28,29,30,31,32,33], while in transplant patients gB1 is the predominant genotype [5,15,16,17,18,19,20,21,22,23,24,25,26,27]. Also, the interpretation of these results is affected by the frequent detection of coinfections with two or more CMV strains in immunosuppressed patients [22,33].…”

“…Among viral factors, genetic variability has been analyzed in order to determine the possible association between viral genetic features and infection outcome; several genes have been targeted for evaluation including those encoding for gB, glycoprotein N, glycoprotein O, and the UL144 gene, among others [6,11,12,13,14]. The gB gene has been studied in different risk groups, including both solid organ and hemopoietic transplant patients [5,15,16,17,18,19,20,21,22,23,24,25,26,27], HIV-infected patients [4,6,15,16,28,29,30,31,32,33], infants with congenital infection [3] and healthy subjects [34]. So far, studies directed towards assessing the impact of genotype features on disease have yielded conflicting results.…”

Mixed infections with distinct cytomegalovirus glycoprotein B genotypes in Polish pregnant women, fetuses, and newborns

“…A Dutch study performed in adults and infants reported the occurrence of double and triple mixed infections composed of various gB genotypes [29]. …”