Herpesvirus-6 Infection Associated Hemophagocytic Lymphohistiocytosis - Excellent Outcome Despite Frequent Central Nervous Involvement: A Case Series Study

Background: Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal condition. Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) overlaps with HLH. Human-herpesvirus 6 (HHV6) is a common childhood infection that rarely causes neurological complications. HHV6-related HLH/DRESS has only been described in case reports . Procedure: From all admissions to Children’s Minnesota with positive HHV6 the records of a subgroup and additional patients from Israel formed a cohort of patients with HHV6-rlated HLH using HLH-2004 criteria. Results: Of 50 consecutive HHV-6 patients, five (10%) developed HLH/DRESS and with two from other centers a cohort of seven is described (six with HLH and one with DRESS). Four had concurrent viruses as possible pathogenic factors. All had thrombocytopenia, elevated soluble IL2 receptor (CD25), and fever; 6/7 had elevated ferritin, and all 5 evaluated had bone marrow hemophagocytosis. Most (6/7) had CNS involvement, all had liver abnormalities, and most had coagulopathy. One patient with Kabuki syndrome was on gammaglobulin replacement therapy, whereas none of the other patients had immune deficiency. One patient had a heterozygous pathogenic variant of TNFSF13B, with no immunodeficiency. Four required etoposide and dexamethasone therapy, while three were also treated with ganciclovir/valganciclovir. The outcome was excellent (median follow-up almost 6 years) with no neurologic sequelae, recurrent HLH, or need for hematopoietic stem cell transplantation. Conclusions: The high incidence of HHV6-related HLH in admitted patients indicates a strong need for vigilance regarding this condition. HHV6-related HLH has a high rate of CNS manifestations, but the outcome in this group of patients was excellent. TITLE PAGE Herpesvirus-6 Infection Associated Hemophagocytic Lymphohistiocytosis - Excellent Outcome Despite Frequent Central Nervous Involvement: A Case Series Study Shifra Ash 1, Yuval Wagner 2,3, Dganit Adam 3,5, Galit Pomeranz Engelberg 3, Joanne Yacobovich 3,4*, Avishalom, Pomeranz 2,3* and Yoav H Messinger 6* 1. Pediatric Hematology-Oncology Department, Ruth Rappaport Children’s Hospital, Rambam Health Care Campus, Haifa 3109601, Israel 2. Pediatric Department, Meir Medical Center, Kfar Saba, Israel 3. School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University 4. Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel 5. Pediatric Intensive Care Unit, Meir Medical Center, Kfar Saba, Israel 6. Department of Hematology-Oncology, Children’s Hospitals of Minnesota, Minneapolis, MN, USA * Joanne Yacobovich, Avishalom Pomeranz and Yoav H Messinger are Co-Senior authors of this manuscript. Corresponding Author: Yoav H Messinger, MD Department of Hematology-Oncology, Children’s Hospitals of Minnesota, 2530 Chicago Av. S, Minneapolis, MN, 55404, USA, Tel +1-612-813-5940, Fax +1-612-813-6325, Email [email protected] Word Count: Abstract 249, Manuscript 3,379 Tables 2, Figures 1, Supporting Information file 1 Short running title: HHV6-HLH case series with CNS manifestations

Human Herpesvirus-6, Hemophagocytic Lymphohistiocytosis, Drug Reaction with Eosinophilia and Systemic Symptoms, Kabuki Syndrome, TNFSF13B variant Published in part as an abstract at The Israeli Society of Pediatric Hematology and Oncology (ISPHO) meeting, Israel, May 2024. Abbreviations Table | CNS | Central Nervous System | | CSA | Cyclosporin-A | | CSF | Cerebrospinal Fluid | | CVID | Common Variable Immunodeficiency | | DRESS | Drug Reaction with Eosinophilia and Systemic Symptoms | | HHV6 | Human Herpesvirus-6 | | HHV7 | Human Herpesvirus-7 | | HLH | Hemophagocytic Lymphohistiocytosis | | HSCT | Hematopoietic Stem Cell Transplantation | | IQ | Interquartile | | IRB | Institutional Review Board | | MOF | Multiorgan Failure | | NK | Natural Killer | | PCR | Polymerase Chain Reaction | | PICU | Pediatric Intensive Care Unit | | PID | Primary Immune Deficiency | | SCID | Severe Combined Immunodeficiency | | sIL2R | Soluble Interleukin-2 Receptor | | TACI | Transmembrane Activator and Cyclophilin Interactor |

Background: Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal condition. Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) overlaps with HLH. Human-herpesvirus 6 (HHV6) is a common childhood infection that rarely causes neurological complications. HHV6-related HLH/DRESS has only been described in case reports . Procedure: From all admissions to Children’s Minnesota with positive HHV6 the records of a subgroup and additional patients from Israel formed a cohort of patients with HHV6-rlated HLH using HLH-2004 criteria.

Of 50 consecutive HHV-6 patients, five (10%) developed HLH/DRESS and with two from other centers a cohort of seven is described (six with HLH and one with DRESS). Four had concurrent viruses as possible pathogenic factors. All had thrombocytopenia, elevated soluble IL2 receptor (CD25), and fever; 6/7 had elevated ferritin, and all 5 evaluated had bone marrow hemophagocytosis. Most (6/7) had CNS involvement, all had liver abnormalities, and most had coagulopathy. One patient with Kabuki syndrome was on gammaglobulin replacement therapy, whereas none of the other patients had immune deficiency. One patient had a heterozygous pathogenic variant of TNFSF13B, with no immunodeficiency. Four required etoposide and dexamethasone therapy, while three were also treated with ganciclovir/valganciclovir. The outcome was excellent (median follow-up almost 6 years) with no neurologic sequelae, recurrent HLH, or need for hematopoietic stem cell transplantation.

The high incidence of HHV6-related HLH in admitted patients indicates a strong need for vigilance regarding this condition. HHV6-related HLH has a high rate of CNS manifestations, but the outcome in this group of patients was excellent. MAIN TEXT

Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and potentially fatal syndrome that is characterized by dysregulation and excessive activation of the immune system. Cytotoxic T cell dysfunction coupled with uninhibited macrophage activity results in overproduction of cytokines and eventually tissue damage. 1 Familial HLH (FHL, genetic, primary) is caused by an underlying genetic defect or complication of other genetic syndromes. 1,2 Secondary or syndromic HLH in children is caused by infections, malignancy, autoimmune or autoinflammatory conditions, and gene therapy in the absence of a known genetic predisposition to HLH. 1-3 HLH has an estimated prevalence of 1 in 100,000 children 4 and familial HLH has a reported incidence of 1.2 in 1,000,000 children. 5 Symptoms and signs of this syndrome include fever, rash, cytopenia, hepatosplenomegaly, coagulopathy, liver dysfunction, and central nervous system (CNS) involvement. 2 Notable laboratory biomarkers include increased ferritin and high soluble interleukin-2 receptor (sIL-2R, CD25) levels as well as elevated cytokines, including CXCL9. The disease can rapidly progress to acute liver failure, multiorgan failure, and death. 2 For clinical studies, the Histiocyte Society developed criteria, the latest of which was used in the HLH-2004 study. 6 These criteria are widely used in clinical practice outside of the realm of clinical trials. 2 Clinical diagnosis of HLH is complicated by an overlap with other syndromes, such as COVID-19 infection and sepsis, and can lead to overdiagnosis or delay in diagnosis, which can result in mortality and morbidity. 2 A modification of nomenclature was suggested by the North American Consortium for Histiocytosis (NACHO) where HLH disease would be categorized according to specific disease contexts instead of the dichotomy of “primary” and “secondary.” 7 For FHL, Henter et al. recently reanalyzed the HLH 2004 diagnostic criteria and concluded that they have excellent accuracy in predicting FHL; however, the use of NK activity is not necessary to achieve significantly high specificity and sensitivity. 8 Human herpesvirus 6 (HHV6) is a common cause of childhood illness, primarily infecting infants, with over 90% of the population infected within the first three years of life. By adulthood, 95% of the population is seropositive for HHV6. 9 The two species of HHV6, HHV6A and HHV6B, infect and establish latency in different cell types, including CD4-T lymphocytes, monocytes, and other epithelial, fibroblastic, and neuronal cells. HHV6A is not associated with any known disease, and its natural history is unknown. In contrast, HHV6B primary infection is ubiquitous in the first two years of life, and subsequent viral latency has the potential for reactivation and disease. 10 HHV6 is the cause of the febrile illness exanthema subitum (also known as roseola infantum), manifesting as high fever followed by a mild skin rash, and is a very common cause of febrile seizures. In approximately 1% of the population, the complete genomes of HHV6A and HHV6B are integrated into the chromosomal telomere and transmitted through Mendelian inheritance. 10 In these patients, persistent viral DNA can be found at very high levels in the blood, cell-free serum, and cerebrospinal fluid (CSF) due to cellular DNA released from damaged cells. 10 HHV6 can cause hepatitis, colitis, gastroenteritis, retinitis, and pneumonitis. 11 Data on the association between HLH and HHV6 is limited to case reports. HLH and HHV6 have been described in the context of primary immune deficiency, with pathogenic variants in IL-2R, 12 XIAP deficiency 13 and isolated CNS HLH. 14 Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) is a syndrome that overlaps with HLH 15 and HHV6 was described in a patient with DRESS secondary to sulfasalazine. 16 It is not known how frequently HLH develops in patients with HHV6. This study aimed to identify a subgroup with HHV6-HLH or HHV6-DRESS from all patients admitted to a tertiary children’s hospital using HHV6 positive quantitative PCR. To further define HHV6-related HLH syndrome, we added two cases of HHV6-HLH from the same era, diagnosed in a primary hospital and transferred to a tertiary hospital to develop a cohort of seven patients (Suppl. Flow diagram). We noted a predominance of CNS manifestations, but excellent prognosis in this group of patients, with no deaths or HLH reactivation.

Children positive for HHV6 by PCR were identified from the data warehouse of Children’s Minnesota, Minneapolis, Minnesota, USA. From the group of HHV6 positive patients five patients were identified as having HLH/DRESS, and their medical records were specifically reviewed. The study was approved by the Institutional Review Board (IRB) of the Children’s Minnesota (IRB# 2024-075). Additionally, we added data on two children with HHV6 induced HLH diagnosed in Meir Medical Center, Kfar Saba, and subsequently treated in Schneider Children’s Medical Center of Israel, Petah Tikva, Israel, who were included in a previously published study. 17 This study was approved by the local ethics committee of Meir Medical Center, Kfar Saba, Israel. All patients with HHV6 quantitative PCR (qPCR) from 2007 to 2020 were included in the first part of the analysis. Quantitative HHV6 PCR was reported for Children’s Minnesota patients by Mayo Laboratory and Quest Diagnostics as copy-number/mL with a negative number of <500 copies/mL. Data extracted from the data warehouse of Children’s Minnesota included baseline characteristics such as age, race/ethnicity, sex, laboratory data, complete blood count (CBC), ferritin and fibrinogen levels (if available), and diagnosis description. It included patients seen in the emergency department or admitted to the hospital. From this cohort, patients with HLH or Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) were reviewed. These patients were designated as CH1, CH2, CH3, CH4, and CH5. Additionally, two patients diagnosed with HHV6-HLH in Meir Medical Center, Kfar Saba, and treated in Schneider Children’s Medical Center of Israel, Petah Tikva, Israel, were designated M2 and M10, and their data is included as previously described. 17 From both the Minnesota group and the group from Israel clinical data included presenting symptoms, laboratory data, length of hospital stay, HLH treatment, and follow-up was collected. For this analysis, HLH was diagnosed according to the Histiocytic Society criteria HLH-2004. 6 Patients who met ≥ 5/8 of the diagnostic criteria were considered positive for HLH. The inclusion criteria were as follows: 1. fever; 2. splenomegaly; 3. Cytopenia of at least two cell lines (bicytopenia): hemoglobin < 10 g/dL, platelets < 100,000/µL, or absolute neutrophil count 265 mg/dL) and/or low fibrinogen levels (500 ng/mL, 6. elevated sIL-2R levels (>2,400 U/mL or > 2 standard deviations from the mean value), 7. hemophagocytosis in the bone marrow, spleen, lymph nodes, or liver; and 8. low/absent NK function. Notably, the CD107a degranulation test 18 failed technically in one patient and was not performed in the others. Perforin expression was evaluated at the Immunology Laboratory at the Cincinnati Children’s Immunology Laboratory. Cytokine levels were available for only one patient, and CXCL9 was not done. T cell immunophenotype analyses were performed by routine flow cytometry, but activated (gaited as HLA-DR+/CD3+) T cells were done evaluated in four Minnesota patients but none of the patients from Israel. When genetic workup was performed, panels specific for HLH-associated genes were used. Qualitative concomitant viral infections were reported by laboratory systems in both hospitals. The outcomes included follow-up at the last visits to Children’s Minnesota (patients CH1-CH5), Schneider Children’s Hematology Clinic, or community clinics (patients M2, M10). Statistical methods: Simple descriptive statistics were provided, but the sample size was too small for statistical significance analysis. HHV6 Cohort From 2007 to 2020, 52 patients from Children’s Minnesota exhibited positive HHV6 PCR results, of whom we excluded 2 family members who were tested to confirm chromosomal integration of HHV6 (Suppl. Flow diagram) to develop a cohort of 50 unique patients described in Table 1. The cohort comprised 58% males and 42% females, with the majority being White/Caucasian (68%), minorities (26%), and unknown ethnicity (6%). The median age was 1.7 years (range, 0 – 25 years; interquartile [IQ] range 1.1 – 7.9 years). Quantitative HHV6 PCR was conducted for all patients, yielding a copy number per mL ranging from 200 to 1,165 – 53,867). Four patients (8%) demonstrated levels copies. Eighteen patients underwent several HHV6 level assessments, with kinetics depicted in Figure 1 with subsequent undetectable levels in 12 of 18 (67%) patients. Two patients with initial HHV6 levels > 2,000,000 for whom follow-up data was available the qPCR remained unchanged in one patient and decreased by three logs in the other patient (Fig. 1). Thirteen patients (26%) required transfer to the pediatric intensive care unit (PICU) with a median length of stay of 7.1 days (IQ range 3.4 – 16.6). Of the 50 patients, three (6%) succumbed to congenital heart disease, failed allogeneic stem cell transplantation for SCIDS, and multiorgan failure; however, none died directly from HLH. The remaining 47 (94%) patients were alive at the last follow-up. HLH Cohort Of the 50 children from Minnesota with positive HHV6 PCR results, five (10%) were diagnosed: four patients with HLH and one patient with DRESS. To allow for a better descriptive analysis, we added two patients with HHV6-HLH diagnosed at Meir Hospital and treated at Schneider Children’s Medical Center of Israel, Israel, for a total of seven patients with HHV6-related HLH. Suppl. Table 1 includes the information for each patient, and Table 2 summarizes the seven patients. This cohort included 5 females and 2 males. Median age 1.75 years (range 0.9-13.4), and 5 (71%) patients were 2,000,000 copies/mL. Two patients had HHV6 chromosomal integration. One of whom (M2) had discrepancy of whole blood to serum HHV6-PCR, the other (CH3) with qPCR whole blood can be vertically transmitted, both the mother and brother of CH3 also exhibit HHV6 chromosomal integration but are not included in Table 1. Four patients (57 %) had concomitant viral infections (HHV7, Human metapneumovirus, adenovirus with rhinovirus, and adenovirus types 4 and 7). Six of the seven patients met the ≥ 5/8 HLH-2004 criteria. Patient CH1 developed carbamazepine-induced DRESS, with a high HHV6 copy number. Although she met the 4/8 HLH-2004 criteria, she had multiple manifestations of HLH, including very high fever, splenomegaly, thrombocytopenia, elevated ferritin, very high IL2R (more than the maximum level for that test), highly elevated activated T-cells 20, severe liver dysfunction, high LDH, coagulopathy, and CNS manifestations with pleocytosis and encephalopathy (Suppl. Table 1). Hemophagocytosis could not be determined because the team decided not to perform a bone marrow evaluation. Thus, we consider this to be a case of DRESS overlapping with HLH. 15 All seven patients had fever, thrombocytopenia, and a high soluble IL2R. Organomegaly was noted in five patients, but only two had splenomegaly. Ferritin was elevated in six patients ranging from 520 to 86,400 ng/dL. Bone marrow was evaluated in five of the patients and all had hemophagocytosis. Natural Killer cell (NK) function was absent or low in four patients and normal in two. Notably, patient CH3 had a low NK function test, later temporarily normalized, and subsequently absent, noting the limitation of that test. The CD107a degranulation test, a better test for HLH, 18 failed in one patient and was not performed in the other patients. Gene panels for HLH were evaluated in five patients, all of whom were originally negative (Suppl. Table 2). However, patent M2 subsequently underwent a full exome study that revealed a heterozygous TNFSF13B c.310T>C p.Cys104Arg pathogenic variant, which was also noted in her asymptomatic mother and sister. Notably, the TNFSF13B variant has not been associated with HLH but is possibly associated with common variable immunodeficiency (CVID). 21 None of her family members (including the patient) have developed signs or symptoms of CVID. Notably, perforin expression was evaluated in five patients, all of whom were normal/high. Activated T cells (HLA-DR+/CD3+), known to be elevated in HLH 20 were found in three of the four patients evaluated, with the highest measured in the DRESS patient (patient CH1). Low IgG at diagnosis was noted in four of the five patients evaluated, although one was a Kabuki Syndrome patient with MLL gene variant on gammaglobulin replacement therapy. A cytokine panel in one patient revealed elevated levels of cytokines typical of HLH (Suppl. Table 1). However, CXCL9 was not done. Critically, 6 of the 7 patients had CNS involvement in HLH, noting seizures and encephalopathy/somnolence, but only one patient had transient changes on MRI. Cerebrospinal fluid (CSF) was evaluated for all five Minnesota patients, revealing pleocytosis in 3 of the 5, elevated protein in 2 of 5, and elevated neopterin in 2 of 3 patients evaluated. All seven patients had liver dysfunction, most severe in one patient (CH4), with metapneumovirus/HHV6 HLH and multiorgan failure (MOF). The majority (six of seven) had significant coagulopathy (three of whom were severe, and 2 had intra-abdominal hemorrhage). All five patients from Minnesota and patient M2 from Israel required transfer to the PICU between 1 and 19 days (Table 2), and MOF was noted in two patients. Therapy consisted of a full HLH-2004 regimen (etoposide, dexamethasone +/- CSA) for four patients (57%) and corticosteroids with/without IVIG in two (29%) patients. The patient with carbamazepine DRESS-HLH recovered after elimination of the offending carbamazepine, but did not require other HLH-directed therapy. Antiviral therapy, including ganciclovir/valganciclovir, was administered to 3 (43%) patients. The outcome was excellent with complete recovery for all patients, no recurrence of HLH, no need for HSCT, no CNS sequalae, and all were alive at a median follow-up of 5.9 years (range 1.5 -12.8 years).

Whether HHV6 was the only viral cause of HLH in six patients or activated DRESS in one patient is not clear. Notably, patient CH2 had HHV7 at a much higher copy number than HHV6, which may have been the trigger for HLH in this patient with Kabuki syndrome undergoing gamma globulin replacement therapy. Other viruses (metapneumovirus and adenovirus) infected the other three patients concurrently with HHV6. Interestingly, a study on malignancy and chemotherapy-related HLH noted six patients (33%) with HHV6, and each patient had concurrent viruses, including EBV. 22 Notably none of our patients had a concomitant EBV infection, which is the most common viral infection associated with HLH. 23 HHV6-related HLH has been reported in patients with primary immune deficiency 12,13 or in patients treated with chemotherapy. 22,24 None of the patients reported by us had received chemotherapy prior to HLH activation. Only one patient in our cohort had an immune deficiency (patient CH2 with Kabuki syndrome and hypogammaglobulinemia on gammaglobulin replacement therapy). Similarly, a patient with Kabuki syndrome developed macrophage activation (MAS), which is equivalent to HLH; however, the patient had no known viral infections. 25 Three of our other patients (CH3, CH5, and M2) had a formal evaluation of immune deficiency that was normal, and the other three showed no symptoms of immune deficiency at follow-up. Thus, our study confirmed that HHV6-related HLH can develop in immunocompetent patients. 26,27 Five patients were evaluated using gene panels specific to HLH, and one of them was later evaluated by whole exome sequencing (Suppl. Table 2 – HLH Genetics). None of the HLH genetic panels was positive; however, the whole exome of patient M2 showed a heterozygous TNFSF13B c.310T>C p.Cys104Arg pathogenic variant. Heterozygote and homozygote variants in TNFRSF13B (transmembrane activator and cyclophilin interactor [TACI] protein) were reported to be associated with common variable immunodeficiency type 2 (CVID 2), 28 but were later considered to only predispose patients to develop CVID. 21 Notably immune function evaluation of M2 with TNFRSF13B was normal (including normal IgA levels), and her sister and mother with the same variant remain asymptomatic. This patient had severe HLH with HHV6 and adenovirus, and required HLH-2004 therapy; thus, this is the first report of HLH in a patient with the TNFRSF13B pathogenic variant. Human herpesvirus 6 (HHV-6) is a neurotropic virus that can cause encephalitis, especially in immunocompromised individuals such as those undergoing hematopoietic stem cell transplantation (HSCT) or organ transplantation. 10,11 HHV-6 establishes latency in various cells, including monocytes, macrophages, and glial cells of the central nervous system (CNS). Immunosuppression, particularly T-cell dysfunction, can lead to reactivation; when HHV6 is reactivated in an immunodeficient host, it is associated with encephalitis. Hematopoietic transplant recipients with resulting T-cell dysfunction can present with post-transplant acute limbic encephalitis (PALE). 10 HHV-6 is one of the most common causes of febrile seizures in infants. 9 Therefore, it is not surprising that six of our seven patients had CNS manifestations of HLH (Table 2). It is well known that CNS-HLH has diverse presentations, can be part of a systemic disease, and can present as an isolated neuroinflammatory disease in familial HLH. 29 Accordingly, our patients with HHV6-HLH and CNS involvement had diverse symptoms, including seizures, abnormal CSF, and severe encephalopathy, but only one brain MRI was abnormal, although some MRI studies were performed late in the disease course. When HHV6-related HLH is complicated by CNS involvement, the reported outcome is very poor, resulting in severe neurological sequelae 30 or several reactivations and ongoing neurological involvement. 14 In contrast, our six patients with HHV6-HLH and CNS involvement (some of whom had severe CNS manifestations) recovered with no known sequelae. Full HLH-2004 therapy, including etoposide and dexamethasone +/- CSA therapy, was used in four of our patients. Antiviral therapy with ganciclovir/valganciclovir, which is known to be effective against HHV6, was used in 3 patients. The outcome of our 7 patients was excellent after a long follow up. Two case reports of single agent therapy steroids +/- ganciclovir/valganciclovir resulted in good outcome. 24,26 In contrast, in another case report single agent steroids therapy resulted in severe neurological sequelae. 30 In contrast to our patient with HHV6 and carbamazepine-DRESS who recovered after withholding the offending agent, a case report of HHV6-related DRESS experienced GI hemorrhage even after withholding the offending agent and died. 16 Most reports of HHV6-related HLH with primary immune deficiency (PID) required HSCT for their PID: a case with XIAP deficiency with HHV6 chromosomal integration and HLH required allogeneic HSCT and prophylactic foscarnet; 13 a case with severe combined immunodeficiency (SCID), osteopetrosis, HHV6 chromosomal integration, and HLH required steroids, HSCT, and prophylactic foscarnet; 12 and a case with FHL3 ( UNC13D variants) required steroids, alemtuzumab, and ganciclovir followed by allogeneic HSCT. 14 In contrast our patient with PID - Kabuki syndrome required full HLH-2004 therapy with excellent long-term outcome and a similar reported case with Kabuki syndrome and MAS responded to corticosteroid, tacrolimus, and hydroxychloroquine sulfate, with good outcome. 25 Thus, therapy for HHV6-related HLH is not currently established and should be based on the general guidelines available to date. 2 The limitations of this study include the retrospective nature of this analysis, and the fact that these patients were diagnosed in an era where some of the more modern assays were not available, including CD107a testing and cytokine panels (including CXCL9). Additionally, due to small numbers we could not determine risk factors for the development of HLH amongst patients with HHV6. As expected, therapy for HLH/DRESS was diverse and the role of anti-viral therapy cannot be clearly defined. However, to the best of our knowledge, this is the only report of a cohort of HHV6-induced HLH cases by the treatment teams. Although conclusions may be limited owing to the small sample size, insights can still be drawn from this analysis. In conclusion, 10% of the consecutively admitted patients with HHV6-positive PCR results developed HLH/DRESS. Of the seven patients described here with HHV6-related HLH/DRESS, CNS involvement, liver dysfunction, and coagulopathy were the most common manifestations. Only one patient had a primary immunodeficiency disorder, and another patient had a pathogenic variant of TNFRSF13B . Four patients required dexamethasone and etoposide therapy, and all recovered with no CNS sequelae, recurrent HLH, or need for HSCT. Therefore, we suggest that HLH should be considered in any patient with HHV6 and significant cytopenia, and that HHV6 should be evaluated in any patient with HLH. Finally, as recommended by Cana et al. 2 any patient with suspected HLH should be evaluated for HLH-related genetic variants, and if negative, more extensive whole genome/exome analysis should be considered, since new genetic variants, such as the TNFRSF13B variant, may be uncovered.

The authors acknowledge the staff at the Cancer and Blood Disorders, Children’s Minnesota, for their great care of patients with HLH. The authors also acknowledge the staff at the PICU, Meir Hospital, and Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center in Israel. Authorship SA, AP, JY, and YHM conceived and designed the study. YHM and his colleagues from Children’s Minnesota were involved with the care of all 5 Minnesota patients. DA and JY managed the patients from Israel. SA, YW, GPE, AP, JY, and YHM abstracted the data and participated in data analysis. SA, JY and YHM drafted the manuscript. All authors have reviewed and approved the manuscript. Funding: This study did not receive any funding. English Language Editing: This manuscript was edited with the help of Paperpal. Disclosure Statement: All authors declare no conflict of interest.

1. Hosahalli Vasanna S, Dalal J. Traffic jam within lymphocytes: A clinician’s perspective. Front Immunol . 2022;13:1034317. 2. Canna SW, Marsh RA. Pediatric hemophagocytic lymphohistiocytosis. Blood . Apr 16 2020;135(16):1332-1343. 3. Galletta F, Cucinotta U, Marseglia L, et al. Hemophagocytic lymphohistiocytosis following gene replacement therapy in a child with type 1 spinal muscular atrophy. J Clin Pharm Ther . Sep 2022;47(9):1478-1481. 4. Niece JA, Rogers ZR, Ahmad N, Langevin AM, McClain KL. Hemophagocytic lymphohistiocytosis in Texas: observations on ethnicity and race. Pediatr Blood Cancer . Mar 2010;54(3):424-8. 5. Henter JI, Elinder G, Söder O, Ost A. Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis. Acta Paediatr Scand . Apr 1991;80(4):428-35. 6. Henter JI, Horne A, Aricó M, et al. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer . Feb 2007;48(2):124-31. 7. Jordan MB, Allen CE, Greenberg J, et al. Challenges in the diagnosis of hemophagocytic lymphohistiocytosis: Recommendations from the North American Consortium for Histiocytosis (NACHO). Pediatr Blood Cancer . Nov 2019;66(11):e27929. 8. Henter JI, Sieni E, Eriksson J, et al. Diagnostic guidelines for familial hemophagocytic lymphohistiocytosis revisited. Blood . Nov 28 2024;144(22):2308-2318. 9. King O, Al Khalili Y. Herpes Virus Type 6. [Updated 2023 Aug 8]. StatPearls [Internet] . 2024 Jan- ed. StatPearls Publishing; 2023. https://www.ncbi.nlm.nih.gov/books/NBK540998/ 10. Ward KN, Hill JA, Hubacek P, et al. Guidelines from the 2017 European Conference on Infections in Leukaemia for management of HHV-6 infection in patients with hematologic malignancies and after hematopoietic stem cell transplantation. Haematologica . Nov 2019;104(11):2155-2163. 11. Agut H, Bonnafous P, Gautheret-Dejean A. Update on infections with human herpesviruses 6A, 6B, and 7. Med Mal Infect . Mar 2017;47(2):83-91. 12. Singh P, Secord E, Pappas K, Savaşan S. An infant with severe combined immunodeficiency, osteopetrosis, chromosomally integrated herpesvirus-6 infection, and hemophagocytic syndrome: What are the links? Pediatr Blood Cancer . Jan 2021;68(1):e28564. 13. Inoue K, Miura H, Hoshino A, et al. Inherited chromosomally integrated human herpesvirus-6 in a patient with XIAP deficiency. Transpl Infect Dis . Oct 2020;22(5):e13331. 14. Bucciol G, Willemyns N, Verhaaren B, et al. Child Neurology: Familial Hemophagocytic Lymphohistiocytosis Underlying Isolated CNS Inflammation. Neurology . Oct 11 2022;99(15):660-664. 15. Yang JJ, Lei DK, Ravi V, Maloney NJ, Crew A, Worswick S. Overlap between hemophagocytic lymphohistiocytosis and drug reaction and eosinophilia with systemic symptoms: a review. Int J Dermatol . Sep 21 2020; 16. Ammar H, Azouzi A, Fathallah N, et al. Fatal sulfasalazine-induced DRESS complicated by HHV-6 reactivation and hemophagocytic lymphohistiocytosis. Eur J Clin Pharmacol . 2020:467-468. vol. 3. 17. Wagner Y, Adam D, Pomeranz Engelberg G, Pomeranz A, Messinger YH. HLH Syndrome in a Community Hospital: The Challenge of an Early Diagnosis. Pediatric Health Med Ther . 2024;15:111-120. 18. Rubin TS, Zhang K, Gifford C, et al. Perforin and CD107a testing is superior to NK cell function testing for screening patients for genetic HLH. Blood . Jun 1 2017;129(22):2993-2999. 19. Pellett PE, Ablashi DV, Ambros PF, et al. Chromosomally integrated human herpesvirus 6: questions and answers. Rev Med Virol . May 2012;22(3):144-55. 20. Chaturvedi V, Marsh RA, Zoref-Lorenz A, et al. T-cell activation profiles distinguish hemophagocytic lymphohistiocytosis and early sepsis. Blood . Apr 29 2021;137(17):2337-2346. 21. Ameratunga R, Woon ST, Gillis D, Koopmans W, Steele R. New diagnostic criteria for common variable immune deficiency (CVID), which may assist with decisions to treat with intravenous or subcutaneous immunoglobulin. Clin Exp Immunol . Nov 2013;174(2):203-11. 22. Strenger V, Merth G, Lackner H, et al. Malignancy and chemotherapy induced haemophagocytic lymphohistiocytosis in children and adolescents-a single centre experience of 20 years. Ann Hematol . Jun 2018;97(6):989-998. 23. Marsh RA. Epstein-Barr Virus and Hemophagocytic Lymphohistiocytosis. Front Immunol . 2017;8:1902. 24. Marabelle A, Bergeron C, Billaud G, Mekki Y, Girard S. Hemophagocytic syndrome revealing primary HHV-6 infection. J Pediatr . Sep 2010;157(3):511. 25. Zhang J, Kang Y, Xia Z, Chong Y, Long X, Shen M. Case report: Macrophage activation syndrome in a patient with Kabuki syndrome. Front Immunol . 2024;15:1412084. 26. Chia XT, Wong HLM, Loh JS. Human herpesvirus-6 infection in a critically ill and immunocompetent patient: a case report. J Med Case Rep . Mar 1 2024;18(1):81. 27. Rahman AU, Baig F, Javid Chaudhary UI, et al. Hemophagocytic Lymphohistiocytosis Associated With Human Herpesvirus-6 (HHV-6) Infection in an Immunocompetent Adult: A Case Report. Cureus . Feb 2024;16(2):e54299. 28. Salzer U, Chapel HM, Webster AD, et al. Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans. Nat Genet . Aug 2005;37(8):820-8. 29. Blincoe A, Heeg M, Campbell PK, et al. Neuroinflammatory Disease as an Isolated Manifestation of Hemophagocytic Lymphohistiocytosis. J Clin Immunol . Aug 2020;40(6):901-916. 30. Tadokoro R, Okumura A, Nakazawa T, et al. Acute encephalopathy with biphasic seizures and late reduced diffusion associated with hemophagocytic syndrome. Brain Dev . Jun 2010;32(6):477-81. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 423views 204downloads Citations Download citation shifra ash, Yuval Wagner, Dganit Adam, et al. Herpesvirus-6 Infection Associated Hemophagocytic Lymphohistiocytosis - Excellent Outcome Despite Frequent Central Nervous Involvement: A Case Series Study. Authorea. 09 January 2025. DOI: https://doi.org/10.22541/au.173641955.58130842/v1 DOI: https://doi.org/10.22541/au.173641955.58130842/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.