Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling

Abstract

Background Human herpesvirus 6 (HHV-6) has a unique ability to integrate into chromosomal telomeres. Vertical transmission via germ cell integration results in offspring with inherited chromosomally integrated (ci)HHV-6 in all nucleated cells, affecting ~1% of the population. Objectives Inherited ciHHV-6 may be a direct or indirect mediator of human disease, but efficient identification of affected individuals is a fundamental roadblock to larger studies exploring the clinical importance of this condition. Study design A group testing strategy was designed to efficiently identify individuals with inherited ciHHV-6. DNA was extracted from 2496 cellular samples from hematopoietic cell transplant (HCT) donor–recipient pairs. Pools of 12 samples were screened for HHV-6 DNA with quantitative (q)PCR. Individual samples from high positive pools were tested with qPCR, and high positive individual samples were tested for inherited ciHHV-6 using droplet digital (dd)PCR to determine HHV-6 DNA copies/cellular genome. Results Thirty-one pools had high positive HHV-6 DNA detection with >103 HHV-6 DNA copies/μg. Each pool had one sample with >104 copies/μg HHV-6 DNA. Inherited ciHHV-6 was confirmed by ddPCR in every high positive sample (>103 HHV-6 DNA copies/μg), yielding a prevalence of 1.5% in HCT recipients and 0.96% in donors. We performed 580 qPCR tests to screen 2496 samples for inherited ciHHV-6, a 77% reduction in testing. Conclusions Inherited ciHHV-6 can be efficiently identified by specimen pooling coupled with modern molecular techniques. This algorithm can be used to facilitate cost-effective identification of patients with inherited ciHHV-6, thereby removing a major hurdle for large-scale study of its clinical impact.

Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6

“…13 We used droplet digital (dd)PCR to determine the ratio of HHV-6 DNA to human cells (using human ribonuclease P DNA [RPP30], a reference gene for cell count). 13,16,19 A ratio of 1 6 0.07 was considered positive for iciHHV-6 and distinguished between iciHHV-6 and naturally acquired infection. This assay was validated against a fluorescence in situ hybridizationconfirmed iciHHV-6 cell line.…”

“…16 This approach leveraged the relatively low population prevalence of iciHHV-6 and high individual viral burden among affected individuals to efficiently identify iciHHV-6. Briefly, approximately 5 mg DNA per sample was aliquoted in 5 mL with the addition of 20 mL buffer AE (Qiagen, CA) (0.2 mg/mL).…”

Regression analysis and variable selection for two‐stage multiple‐infection group testing data

“…6 Since this seminal work, pooling techniques have been successfully implemented to screen for many other infectious diseases, including HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), 7 influenza, 8 and herpes. 9 Besides disease screening, many other areas, including genetics, 10 veterinary science, 11 medical entomology, 12 blood safety, 13 and drug discovery, 14 have also used the method of pooling. Statistical research in group testing primarily focused on improving the diagnostic accuracy and cost-saving ability of a pooling protocol 15 or estimating individual-level characteristics from pooled testing data.…”

“…Pooled testing (also known as group testing) was initially proposed to screen for syphilis among War World II American army recruits . Since this seminal work, pooling techniques have been successfully implemented to screen for many other infectious diseases, including HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), influenza, and herpes . Besides disease screening, many other areas, including genetics, veterinary science, medical entomology, blood safety, and drug discovery, have also used the method of pooling.…”

Multiple transmissions of chromosomally integrated human herpesvirus‐6 in one family

“…In addition, the rise in HHV‐6 copies paralleled engraftment and rising WBC. A diagnosis of ciHHV‐6 was confirmed by a whole blood quantitative droplet digital PCR assay (ddPCR; Molecular Virology, University of Washington, Seattle, WA, USA) on Day +27 of the second syngeneic HCT using a previously described assay . Briefly, ddPCR provides a precise calculation of the number of HHV‐6 DNA copies per cell; this ratio is expected to be 1 in patients with ciHHV‐6.…”