Investigators at University of Washington studied multiple samples to develop an assay that can detect active infection in patients with chromosomally integrated HHV-6. Current quantitative PCR DNA testing cannot determine whether a ciHHV6 patient has active replication.
Serge Barcy, PhD, Seattle Children’s Research Institute, Seattle, Washington
A team led by Serge Barcy, PhD and Joshua Hill, MD at University of Washington performed RNA sequencing on multiple sample types to characterize the HHV-6B transcriptome, and found that U38 transcripts appear to be a sensitive and specific target for identifying active HHV-6B infection. Transplant physicians treating individuals impacted by ciHHV-6 are unable to determine whether these patients require antiviral therapy.
Samples were collected from several sources:
- Whole blood from hematopoietic stem cell transplant (HCT) recipients
- Tumor tissue samples from patients with large B cell lymphoma infected with HHV-6B
- Lymphoblastoid cell lines from patients with inherited chromosomally integrated HHV-6B (ciHHV-6B) or latent infection with HHV-6B
- HHV-6B infected SupT1 CD4+ T cells
There was substantial overlap in the HHV-6B transcriptome between the samples, although the only HHV-6B transcript detected in all RNA-seq data sets was the HHV-6B viral polymerase gene, U38. As a result, they developed a novel reverse transcription qPCR assay (RT-qPCR) targeting the U38 gene, which identified U38 mRNA in all tested whole blood samples from patients with concurrent HHV-6B viremia. No U38 mRNA was detected by RNA-seq or RT-qPCR in whole blood samples from subjects without HHV-6B DNA positivity in plasma or from latently infected lymphoblastoid cells.
In this study, Hill et al. utilized whole blood collected in PAXgene Blood RNA tubes within the first six weeks of HCT as part of another unrelated study. Then they identified plasma samples from these subjects that were collected before, on the day of, or after the PAXgene tube collection. Patients who had HHV-6B+ plasma samples within 7 days of PAXgene tubes were selected for this study and those without HHV-6B detection within 7 days of PAXgene tube collection were used as controls. Patients were matched 1:1 based on an array of criteria including the day of PAXgene tube collection and type of HCT. Additionally, HHV-6B Z29-infected SupT1 CD4+ T cells provided by the HHV-6 Foundation and data from two patients with DLBCL from a study in which RNA-seq on tumor tissue samples demonstrated a broad profile of HHV-6B transcripts consistent with lytic replication served as additional controls.
There were seven total subjects who had concurrent HHV-6B DNA detection in plasma with a median of 4 days between plasma detection and PAXgene tube collection. Matched controls were identified for the first five cases. None of the subjects developed HHV-6-associated end-organ disease. Sufficient mRNA was isolated for 5/7 cases and 3/5 controls. The lowest yields of total RNA were observed for subjects with the lowest absolute WBC counts. HHV-6B gene expression was identified in 4/5 cases and 0/3 controls. There was no evidence of correlation between plasma HHV-6B DNA loads and total HHV-6B gene transcript counts in whole blood samples detected by RNA-seq. Overall, the whole blood samples had the lowest HHV-6B gene counts (range 0.03-19 VPMM), whereas the 2 DLBCL tumor tissue samples had higher gene counts (21 and 94 VPMM) with the in vitro SupT1 culture displaying the highest gene count (7,040 VPMM).
Only the U38 gene was detected in all sample types, thus it was selected as the diagnostic target for RT-qPCR development. The sensitivity of the assay was validated using mRNA extracted from whole blood in 4 of the post-HCT cases with concurrent HHV-6B positivity in plasma. The U38 gene was detected in all four cases; additionally, the high sensitivity of the assay was further demonstrated by the detection of U38 mRNA in one subject in which RNA-seq did not identify any HHV-6B gene transcripts as well as another case in which there was insufficient amount of isolated RNA to perform RNA-seq. The number of U38 copies detected by RT-qPCR correlated with the number of reads mapping to U38 by RNA-seq (r2 =1.0; p=0.08). Statistical significance was not reached likely due to small sample size.
The specificity of this assay was demonstrated using whole blood from all 5 post-HCT controls who did not have HHV-6B+ positive plasma, as well as the 2 lymphoblastoid cell lines with latent HHV-6 that did not have HHV-6B gene transcript reads identified by RNA-seq. U38 was not detected in any of these samples, whereas GAPDH was detected by RT-qPCR in the aforementioned samples, indicating sufficient RNA isolation. Overall, these data suggest that this U38 RT-qPCR assay displays ample sensitivity and specificity for further development as a clinical diagnostic assay.
Limitations of this study include a small sample size, a lack of longitudinally obtained samples (which would have allowed analysis of temporal HHV-6B gene expression patterns), and a lack of subjects who had severe HHV-6 infection with end-organ disease.
Unfortunately, plasma qPCR on plasma samples, the current “gold standard” for HHV-6 testing, cannot identify active HHV-6 infection in organs as HHV-6 infection is often highly localized with no detectable plasma viremia (Leveque 2011, Buyse 2013, Caserta 2011, Phan 2018). Post-transplant DNAemia is also often subclinical.
Future studies using sequentially collected samples from before and after HHV-6B reactivation (which should include those with ciHHV-6) are needed to validate and refine the findings in this study.
Read the paper here: Hill 2018