Joshua A. Hill

Coreactivation of human herpesvirus 6 and cytomegalovirus is associated with worse clinical outcome in critically ill adults

Objectives:Human herpesvirus 6 is associated with a variety of complications in immunocompromised patients, but no studies have systematically and comprehensively assessed the impact of human herpesvirus 6 reactivation, and its interaction with cytomegalovirus, in ICU patients. Design:We prospectively assessed human herpesvirus 6 and cytomegalovirus viremia by twice-weekly plasma polymerase chain reaction in a longitudinal cohort study of 115 adult, immunocompetent ICU patients. The association of human herpesvirus 6 and cytomegalovirus reactivation with death or continued hospitalization by day 30 (primary endpoint) was assessed by multivariable logistic regression analyses. Setting:This study was performed in trauma, medical, surgical, and cardiac ICUs at two separate hospitals of a large tertiary care academic medical center. Patients:A total of 115 cytomegalovirus seropositive, immunocompetent adults with critical illness were enrolled in this study. Interventions:None. Measurements and Main Results:Human herpesvirus 6 viremia occurred in 23% of patients at a median of 10 days. Human herpesvirus 6B was the species detected in eight samples available for testing. Most patients with human herpesvirus 6 reactivation also reactivated cytomegalovirus (70%). Severity of illness was not associated with viral reactivation. Mechanical ventilation, burn ICU, major infection, human herpesvirus 6 reactivation, and cytomegalovirus reactivation were associated with the primary endpoint in unadjusted analyses. In a multivariable model adjusting for mechanical ventilation and ICU type, only coreactivation of human herpesvirus 6 and cytomegalovirus was significantly associated with the primary endpoint (adjusted odds ratio, 7.5; 95% CI, 1.9–29.9; p = 0.005) compared to patients with only human herpesvirus 6, only cytomegalovirus, or no viral reactivation. Conclusions:Coreactivation of both human herpesvirus 6 and cytomegalovirus in ICU patients is associated with worse outcome than reactivation of either virus alone. Future studies should define the underlying mechanism(s) and determine whether prevention or treatment of viral reactivation improves clinical outcome.

Detection of Human Herpesvirus 6B (HHV-6B) Reactivation in Hematopoietic Cell Transplant Recipients with Inherited Chromosomally Integrated HHV-6A by Droplet Digital PCR

ABSTRACT The presence of inherited chromosomally integrated human herpesvirus 6 (ciHHV-6) in hematopoietic cell transplant (HCT) donors or recipients confounds molecular testing for HHV-6 reactivation, which occurs in 30 to 50% of transplants. Here we describe a multiplex droplet digital PCR clinical diagnostic assay that concurrently distinguishes between HHV-6 species (A or B) and identifies inherited ciHHV-6. By applying this assay to recipient post-HCT plasma and serum samples, we demonstrated reactivation of HHV-6B in 25% (4/16 recipients) of HCT recipients with donor- or recipient-derived inherited ciHHV-6A, underscoring the need for diagnostic testing for HHV-6 infection even in the presence of ciHHV-6.

Hepatitis due to human herpesvirus 6B after hematopoietic cell transplantation and a review of the literature.

Human herpesvirus 6B (HHV‐6B) is an opportunistic pathogen associated with a growing number of complications in immunocompromised patients. Multiple reports of HHV‐6B‐associated hepatitis following primary HHV‐6 infection and liver transplantation have appeared, but this has only been well documented in 1 patient after hematopoietic cell transplantation (HCT). This report describes a case of acute hepatitis likely caused by HHV‐6B in an HCT recipient who was successfully treated with ganciclovir. HHV‐6B DNA was demonstrated in plasma and hepatic tissue using quantitative polymerase chain reaction and immunohistochemical stains. Chromosomal integration was ruled out. We review the literature reporting HHV‐6B‐associated hepatitis, which may be an underappreciated cause of liver disease after HCT.

Human herpesvirus 6B reactivation and delirium are frequent and associated events after cord blood transplantation

Human herpesvirus 6B (HHV-6B) frequently reactivates after cord blood transplantation (CBT). We previously reported an association between HHV-6B reactivation and delirium after hematopoietic cell transplantation. In this prospective study, 35 CBT recipients underwent twice-weekly plasma PCR testing for HHV-6 and thrice-weekly delirium assessment until day 84. There was a quantitative association between HHV-6B reactivation and delirium in univariable (odds ratio, 2.88; 95% confidence interval (CI), 0.97–8.59) and bivariable models. In addition, intensified prophylaxis with high-dose valacyclovir mitigated HHV-6B reactivation (adjusted hazard ratio, 0.39; 95% CI, 0.14–1.08). Larger trials are needed to explore the utility of HHV-6B prophylaxis after CBT.

RNA sequencing of the in vivo human herpesvirus 6B transcriptome to identify targets for clinical assays distinguishing between latent and active infections

Human herpesvirus 6B (HHV-6B) DNA is frequently detected in human samples, especially after hematopoietic cell transplantation (HCT). Diagnostic assays distinguishing HHV-6B reactivation from latency are limited, and this has contributed to confusion in research and made the design of clinical approaches to diagnose and treat HHV-6-associated diseases challenging. We used RNA sequencing to characterize and compare the HHV-6B transcriptome in multiple in vivo and in vitro sample types, including 1) whole blood from HCT recipients with and without HHV-6B plasma viremia; 2) tumor tissue samples from subjects with large B cell lymphoma infected with HHV-6B; 3) lymphoblastoid cell lines from subjects with inherited chromosomally integrated HHV-6B or latent infection with HHV-6B; and 4) HHV-6B Z29 infected SupT1 CD4+ T cells. We demonstrated substantial overlap in the HHV-6B transcriptome observed in in vivo and in vitro samples, although there was variability in the breadth and quantity of gene expression across samples. No HHV-6B transcripts were detected in whole blood samples from subjects without plasma HHV-6B viremia. The HHV-6B viral polymerase gene U38 was the only HHV-6B transcript detected in all RNA-seq data sets and was one of the most highly expressed genes. Using a novel reverse transcription PCR assay targeting HHV-6B U38, we identified U38 messenger RNA in all tested whole blood samples from patients with concurrent HHV-6B viremia, indicating its utility as a diagnostic assay for HHV-6B replication. This study demonstrates the feasibility of pathogen transcriptome analyses in HCT recipients to identify better targets for diagnostic, and potentially therapeutic, applications. IMPORTANCE Infection with human herpesvirus 6B (HHV-6B), a DNA virus, occurs early in life, results in chronic viral latency in diverse cell types, and affects the population at large. Additionally, HHV- 6B can integrate into germline chromosomes, resulting in individuals with viral DNA in every nucleated cell. Given that PCR to detect viral DNA is the mainstay for diagnosing HHV-6B infection, the characteristics of HHV-6B infection complicate efforts to distinguish between latent and active viral infection, particularly in immunocompromised patients who have frequent HHV- 6B reactivation. In this study, we used RNA sequencing to characterize the HHV-6B gene expression profile in multiple sample types, and our findings identified evidence-based targets for diagnostic tests that distinguish between latent and active viral infection.

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

Chromosomally integrated human herpesvirus‐6 (ciHHV‐6) can be transmitted from parent to child or via allogeneic hematopoietic cell transplantation (HCT). We report a case of ciHHV‐6 transmitted via syngeneic HCT, and vertically across 3 generations. ciHHV‐6 was transmitted from a parent to the patient and her identical twin, and from the patient to her son. The patient underwent syngeneic HCT as rescue from chemotherapy‐induced aplasia during which ciHHV‐6 was re‐transmitted to her, this time from her identical twin. This is the first report, to our knowledge, of a patient acquiring ciHHV‐6 once via germline from a parent and again via syngeneic HCT from an identical twin.

Alveolar levels of immuno-inflammatory mediators in diffuse alveolar hemorrhage after allogeneic transplant

Idiopathic pneumonia syndrome (IPS) is a severe and morbid complication of hematopoietic cell transplantation (HCT) that largely lacks effective treatments. The syndrome is defined as abnormal pulmonary physiology with multilobar opacities on chest imaging that are not attributable to lower respiratory tract infection (LRTI) or other organ failures [1]. Many pathologies meet IPS criteria, including organizing pneumonia, interstitial pneumonitis, and diffuse alveolar damage, among others. Biologic heterogeneity within this broadly defined syndrome may diminish the ability to detect the effect of treatments. An improved understanding of IPS pathobiology may catalyze the development of effective preventive and therapeutic strategies [2]. Several pro-inflammatory cytokines/chemokines are elevated in IPS [3, 4]. These biomarkers may be useful to distinguish IPS subphenotypes with unique biologic features. As a preliminary examination of this concept, we tested the hypothesis that immuno-inflammatory mediators measured in the blood and alveolar compartments of people with IPS differ according to the presence or absence of diffuse alveolar hemorrhage (DAH). DAH is a variably present manifestation of IPS that is proposed to have distinct biology [5]. We retrospectively studied 17 adults who underwent clinical bronchoscopy for diffusely abnormal chest imaging within 120 days of allogeneic HCT at Fred Hutchinson Cancer Research Center (FHCRC) between 2008 and 2010. None received specific tumor necrosis factor (TNF)-α inhibitors prior to bronchoscopy. Data and samples were banked prospectively with subjects’ consent under FHCRC protocol 999.209 and were approved for use in this research (FHCRC protocols 808 and 1829/substudy 050). We reviewed pertinent progress notes, microbiology reports, pathology reports, bronchoscopy reports, and radiographic images to identify IPS cases according to the American Thoracic Society definition [1]. We defined abnormal respiratory physiology as oxygen saturation ≤92% by pulse oximetry or arterial blood gas while breathing ambient air or supplemental oxygen. We defined LRTI as bronchoalveolar lavage fluid (BALF) growing any amount of Gram-negative rods, Mycoplasma spp., Chlamydophila spp., fungi/mold, Legionella, Nocardia, Mycobacteria, or >10 colony-forming units per mL of a single pathogenic Gram-positive coccus. Cytomegalovirus (CMV) infection required both culture and PCR positivity. We also classified as LRTI: any positive direct fluorescence antibody stain or >40 PCR copies of respiratory viruses (such as adenovirus, coronavirus, parainfluenza, influenza A/B, respiratory syncytial virus, human metapneumovirus, herpes simplex, and varicella zoster); positive stain for Pneumocystis jirovecii; and BALF or serum galactomannan >0.5 ng/L. DAH required documentation of increasingly bloody return from serial bronchoalveolar lavages, or report of “bloody” BALF followed by treatment with systemic corticosteroids for clinically diagnosed DAH. According to local protocols, blood samples were drawn at weekly intervals to survey for CMV and processed and cryopreserved immediately using local standard operating procedures. When available, we selected blood samples collected prior to bronchoscopy or within 2 days of bronchoscopy to best reflect biologic activity at the time of bronchoscopy. BALF samples were obtained using standard clinical techniques and cryopresevered without processing. We thawed samples on an ice batch and centrifuged them at * Lisa K. Vande Vusse lkvandev@u.washington.edu