Acyclovir‐Resistant Herpes Simplex Virus in Pediatric Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant: A Case Series

ABSTRACT

Background

Herpes simplex virus (HSV) infection is a significant risk in hematopoietic stem cell transplant (HSCT) recipients, and antiviral resistance poses many clinical challenges.

Methods

A retrospective case series of eight pediatric patients with acyclovir‐resistant HSV infection, determined via genotypic sequencing, post allogeneic HSCT between 2012 and 2025.

Results

Indications for HSCT included primary immunodeficiency, sickle cell disease, myelodysplastic syndrome (MDS), and relapsed/refractory leukemia. All patients received acyclovir prophylaxis. TK mutations were most common (n = 7, 87.5%). Infections manifested as mucocutaneous disease, pneumonitis, keratitis, enterocolitis, and blood viremia. A total of 87.5% of patients (n = 7) were treated with second‐line antivirals (foscarnet and/or cidofovir), and three patients were treated with third‐line antivirals (pritelivir). Treatment was complicated by nephrotoxicity (n = 3, 37.5%). Adjunct treatments included intravenous immunoglobulin (n = 3, 37.5%) and donor lymphocyte infusion (n = 1, 12.5%). Poor outcomes were seen across the cohort, including death (n = 3, 37.5%). T‐cell depletion was used in 50% of patients (n = 4). Concomitant immunosuppressive therapy was used in 100% of patients (n = 8) and high‐dose steroids were used in 50% of patients (n = 4). Graft versus host disease occurred in four patients (50%). Secondary complications included transplant‐associated thrombotic microangiopathy (n = 3, 37.5%), idiopathic pulmonary syndrome (n = 3, 37.5%), and graft failure (n = 1, 12.5%).

Conclusion

Acyclovir‐resistant HSV infection in pediatric HSCT recipients is associated with high morbidity and mortality, limited therapeutic options, and significant treatment‐related nephrotoxicity. Early recognition, early resistance testing, prompt initiation of second‐line therapy, and weaning of immunosuppression are critical. Emerging therapies, such as helicase‐primase inhibitors and adoptive T‐cell therapy, hold promise but remain limited by access and pediatric data.

This retrospective case series of acyclovir‐resistant HSV infection in pediatric patients undergoing allogeneic HSCT discusses the clinical course, associated complications, and treatment, including side effects. We propose the first algorithm for investigating and managing acyclovir‐resistant HSV infections in pediatric HSCT recipients, highlighting the need for early recognition, early resistance testing, prompt initiation of second‐line therapy, and weaning of immunosuppression, in order to control disease.

Abbreviations

ATG

antithymocyte globulins

CMV

cytomegalovirus

DLI

donor lymphocyte infusion

DNA

deoxyribonucleic acid

EBV

Epstein–Barr virus

GVHD

graft versus host disease

HHV6

human herpes virus 6

HPI

helicase‐primase inhibitor

HSCT

hematopoietic stem cell transplant

HSV

herpes simplex virus

HSV‐1

herpes simplex virus type 1

HSV‐2

herpes simplex virus type 2

IPS

idiopathic pulmonary syndrome

IvIg

intravenous immunoglobulin

MDS

myelodysplastic syndrome

MSD

matched sibling donor

MUD

matched unrelated donor

PCR

polymerase chain reaction

TA‐TMA

transplant‐associated thrombotic microangiopathy

TK

thymidine kinase

UCB

unrelated cord blood

1. Introduction

Herpes simplex viruses (HSV) are double‐stranded deoxyribonucleic acid (DNA) viruses, which belong to the Herpesviridae family [1, 2]. The virus is known for its ability to remain in a latent state within dorsal root ganglia and the autonomic nervous system, allowing future reactivations of the disease [1, 2]. HSV reactivation or primary infection in immunocompromised patients can be caused by either HSV type 1 (HSV‐1) or HSV type 2 (HSV‐2) [2]. Typically, the disease manifests as isolated mucocutaneous disease in the orofacial region, but less frequent manifestations include pneumonitis, hepatitis, meningitis (HSV‐2), and encephalitis (HSV‐1) [2]. In immunocompromised patients, HSV infection is associated with greater severity of clinical disease, risk of hematogenous dissemination, and increased risk of selecting mutants resistant to antiviral treatments [2, 3]. Patients undergoing hematopoietic stem cell transplant (HSCT) receive systemic antiviral prophylaxis, which has reduced the incidence of HSV infection in this cohort from 80% to 10% [2, 4]. It is unclear whether prolonged exposure to prophylactic acyclovir increases the risk of developing resistance [5]. Acyclovir is used for both prophylaxis and treatment of HSV infections in HSCT recipients. Acyclovir is a nucleoside analogue drug, which requires phosphorylation by HSV‐encoded thymidine kinase (TK) [6]. The phosphorylated form acts as a competitive inhibitor of DNA polymerase, such that incorporation of the phosphorylated form into the replicating viral DNA results in premature termination of the viral DNA chain, thereby preventing viral replication [6]. Resistance to acyclovir occurs either via mutations involving TK or DNA polymerase [6]. TK mutations in the UL23 gene make up 95% of resistance mutations and lead to resistance to acyclovir, but spare foscarnet and cidofovir, whose activation does not require phosphorylation [2, 6]. In contrast, mutations in DNA polymerase involving the UL30 gene, which make up 5% of known mutations, confer resistance to acyclovir but also to foscarnet and sometimes cidofovir [2]. Resistance testing can be done via phenotypic or genotypic assay [7], but consensus guidelines for HSCT recipients do not currently exist.

This paper discusses eight cases of acyclovir‐resistant HSV in pediatric patients undergoing allogeneic HSCT, the largest pediatric series to date. This case series aims to describe the clinical course, associated complications, and treatment course of these patients, including side effects. To date, there is no consensus guideline on the investigation and treatment of acyclovir‐resistant HSV infections in HSCT recipients; herein we propose the first algorithm for pediatric allogenic HSCT recipients.

2. Methods

Acyclovir resistance was determined using genotypic nucleotide sequencing following polymerase chain reaction (PCR) amplification of gene targets. Gene targets included TK gene (UL23), DNA polymerase (UL30), and DNA helicase (UL5). Patients were eligible for inclusion if they had either primary HSV infection or HSV reactivation.

2.1. Clinical Cases

A total of eight patients were identified with acyclovir‐resistant HSV at Royal Manchester Children's Hospital between 2012 and 2025. A summary of the clinical cases is shown in Table 1 (see Appendix S1 for detailed case summaries). Patients were aged between 1.5 and 17 years old and included 62.5% males (n = 5) and 37.5% females (n = 3). Indications for HSCT included primary immunodeficiency, sickle cell disease, myelodysplastic syndrome (MDS), and relapsed/refractory leukemia. Donor sources included unrelated cord blood (UCB), matched unrelated donor (MUD), matched sibling donor (MSD), and haploidentical donor. T‐cell depletion was used in four patients (n = 50%). All patients were on immunosuppression at the time of HSV infection and all received antiviral prophylaxis with acyclovir.

TABLE 1.

Patient and clinical characteristics of acyclovir‐resistant HSV infections.

Case	Age (years)	Diagnosis	HSCT no.	Year	Donor	Conditioning	Immuno‐suppression a	Duration prior ACV	Onset of HSV	Clinical manifestations of HSV	Mutation identified	Antiviral drugs used	Immune recovery b	Outcome of HSV infection
1	1.5	JMML, secondary graft failure post first HSCT	2	2025	8/8 UCBT	FTT	CsA MMF Methylprednisolone	4 months	D+24	Disseminated disease—mucocutaneous, enterocolitis, pneumonitis, blood viremia Secondary—TA‐TMA, IPS	TK 460‐464delC	Acyclovir Foscarnet Pritelivir	ALC = 1.5 × 109/L CD4 = 138 cells/µL CD3 = 503 cells/µL CD8 = 354 cells/µL	Non‐responder ‐Died secondary to severe HSV‐associated ARDS.
2	17	Refractory un‐differentiated leukemia	1	2023	6/8 UCBT	Flu/Treo	CsA MMF Methylprednisolone	1 month	D+18	Mucocutaneous, blood viremia, pneumonitis	TK S181N	Valacyclovir Foscarnet	ALC = 0.8 × 109/L CD4 = 194 cells/µL CD3 = 460 cells/µL CD8 = 257 cells/µL	Responder ‐Blood and mucocutaneous (5 days foscarnet) ‐Respiratory (7 days foscarnet)
3	8	Relapsed T‐ALL	1	2025	7/8 UCBT	TBI/etoposide	CSA MMF	1 month	D+17	Mucocutaneous	TK 548‐553insC	Acyclovir	ALC = 0.4 × 109/L CD4 = 286 cells/µL CD3 = 353 cells/µL CD8 = 70 cells/µL	Responder ‐Resolution 19 days after onset
4	15	Sickle cell disease with CNS vasculopathy, primary graft failure	2	2024	Haplo	Flu/Cy alemtuzumab	Sirolimus, MMF, abatacept	2 months	D+18	Mucocutaneous, blood viremia	TK novel W88 stop codon Novel A904S	Acyclovir Foscarnet Pritelivir	ALC = 0.62 × 109/L CD4 = 478 cells/µL CD3 = 530 cells/µL CD8 = 45 cells/µL	Responder ‐Blood viremia (2 days foscarnet) ‐Mucocutaneous (10 days foscarnet)
5	1.5	MDS with SAMD9 and monosomy 7	1	2024	10/10 MUD	FTT alemtuzumab	CsA Methylprednisolone	2 months	First—D+44 Second—D+159	Disseminated disease—mucocutaneous lesions, blood viremia, pneumonitis Secondary—TA‐TMA, graft failure, IPS	TK 430‐436insG DNA Pol R700G	Acyclovir Foscarnet Pritleivir	ALC = 0.1 × 109/L CD4 = 293 cells/µL CD3 = 356 cells/µL CD8 = 38 cells/µL ———– ALC = <0.1 × 109/L CD4 = 5 cells/µL CD3 = 98 cells/µL CD8 = 76 cells/µL	First responder ‐Blood viremia (7 days foscarnet) ‐Mucocutaneous (28 days pritelivir) ‐Respiratory viremia (24 days pritelivir) Second non‐responder ‐Died secondary to refractory HSV, severe ARDS
6	15	Refractory B‐ALL	1	2022	10/10 MUD	TBI/etoposide alemtuzumab	CsA MMF	1 month	D+26	Pneumonitis Secondary—TA‐TMA, IPS	TK 430‐436insG TK A93V	Acyclovir Foscarnet	ALC = 0.1 × 109/L	Non‐responder ‐Patient died of multi‐organ failure secondary to HSV
7	15	Refractory secondary AML post first HSCT	2	2021	7/8 UCBT	Flu, Bu	CsA MMF Methylprednisolone Ruxolitinib	4 months	D−1	Mucocutaneous lesions, herpetic keratitis, blood viremia	TK430‐436insG	Cidofovir Foscarnet	ALC = 0 × 109/L	Indeterminate ‐Patient died of multi‐organ failure
8	17	T‐ALL CNS relapse in remission. Late aplastic graft failure after previous HSCT	2	2025	9/10 MUD	Flu/Cy alemtuzumab	CsA MMF	6 months	D−7	Mucocutaneous, blood viremia	Mutation of unknown effect I619N	Acyclovir foscarnet	ALC = < 0.1 × 109/L CD4 = 7 cells/µL CD3 = 7 cells/µL CD8 = 3 cells/µL	Responder ‐Blood viremia (5 days foscarnet) ‐Mucocutaneous (13 days foscarnet)

Abbreviations: A = acyclovir; ALC = absolute lymphocyte count; ALL = acute lymphoblastic leukemia; ARDS = acute respiratory distress syndrome; BAL = bronchioalveolar lavage; BU = busulfan; CsA = ciclosporin; DLI = donor lymphocyte infusion; DNA Pol = DNA polymerase; Flu/Cy = fludarabine, cyclophosphamide; Flu/Treo = fludarabine, treosulfan; FTT = fludarabine, treosulfan, thiotepa; Haplo = haploidentical donor; HFOV = high frequency oscillatory ventilation; HSV = herpes simplex virus; IvIg = intravenous immunoglobulin; JMML = juvenile myelomonocytic leukemia; MMF = mycophenolate mofetil; MUD = matched un‐related donor; PICU = pediatric intensive care unit; TA‐TMA = transplant‐associated thrombotic microangiopathy; TBI = total body irradiation; TK = thymidine kinase; UCBT = un‐related cord blood transplant.

^a Immunosuppression at the time of HSV infection.

^b Immune recovery at time of HSV clearance.

Resistance is thought to have developed during prophylaxis as patients did not respond to first‐line treatment started at the time of HSV infection and had no prior history of antiviral treatment. Duration of acyclovir prophylaxis varied (1–6 months) and did not correlate with resistance. All patients isolated HSV‐1 strain. TK mutations were the most common identified (n = 7, 77%), with DNA polymerase mutations less frequently detected (n = 1, 11%). Clinical manifestations included mucocutaneous disease, pneumonitis, herpetic keratitis, enterocolitis, and blood viremia. Note that, 87.5% of patients (n = 7) were treated with second‐line antivirals, and 37.5% (n = 3) were treated with third‐line antivirals. Patients were weaned off immunosuppression to aid immune reconstitution. Adjunct treatments used included intravenous immunoglobulin (IvIg, n = 3, 33%) and donor lymphocyte infusion (DLI, n = 1, 11%). Poor outcomes were seen across the cohort, with three deaths (37.5%) attributed to HSV infection and its complications. Graft versus host disease (GVHD) occurred concomitantly in four patients (50%). Secondary complications were observed in three patients (37.5%) and included transplant‐associated thrombotic microangiopathy (TA‐TMA, n = 3, 37.5%), idiopathic pulmonary syndrome (IPS, n = 3, 37.5%), and graft failure (n = 1, 12.5%).

3. Discussion

This case series discusses eight pediatric patients post allogeneic HSCT with acyclovir‐resistant HSV infections and highlights the significant morbidity and mortality associated with these infections and the large unmet need for safe and effective treatments in this cohort.

While significance cannot be determined due to low patient numbers and insufficient statistical power, several potential risk factors for acyclovir‐resistant HSV emerged from reviewing the cases in our center. Recurrent factors associated with acyclovir‐resistance HSV infection included high‐risk disease as indication for transplant, with 62.5% of patients (n = 5) having relapsed/refractory leukemia and 50% of the cohort (n = 4) undergoing their second HSCT. This finding is consistent with the literature, which has reported HSCT for relapsed hematologic malignancy as a risk factor for acyclovir‐resistant HSV infections [2, 5, 8, 9]. If we consider the donor used, HLA mismatch was seen in 62.5% of our cohort (n = 5), which has been previously reported as a risk factor [2, 5, 8, 9]. Immunosuppression was also a common factor, with T‐cell depletion used in 50% of the cohort (n = 4), concomitant immunosuppressive therapy in 100% of patients (n = 8), and concomitant use of high‐dose steroids in 50% of patients (n = 4). In addition, 87.5% of patients (n = 7) had poor immune reconstitution at the time of infection. While use of antithymocyte globulin (ATG) and corticosteroids has been reported as potential risk factors [2], T‐cell depletion and poor immune reconstitution have not previously been reported. The mechanism by which use of immunosuppression and poor immune reconstitution predisposes to acyclovir resistance may be due to the impaired host immune response resulting in defective HSV clearance, thus enabling ongoing viral replication [8]. In the presence of prophylactic acyclovir, ongoing viral replication may increase selection pressure and the emergence of resistant strains [8]. Concomitant GVHD was also seen in 50% of our cohort, and Grade ≥ 2 GVHD has been previously reported in the literature to be associated with acyclovir‐resistant HSV infections [2, 5, 8, 9], presumably due to increased use of immunosuppression.

One of the problems highlighted by our case series was the ineffectiveness of current treatments in managing resistant HSV infections. TK mutations were the most common mutation identified in our cohort (87.5%, n = 7), and as mentioned previously, TK mutations lead to resistance to acyclovir, but not to foscarnet or cidofovir [2, 6]. Despite this, of the seven patients in this cohort with TK mutations, only three patients had effective treatment and clearance of their HSV infection with either foscarnet and/or cidofovir. In addition, for the three patients who were treated with third‐line antivirals, only one had a good clinical response with resolution of their infection. Within the cohort, despite utilization of second‐ and third‐line antiviral therapy, patients continued to experience severe infections, with mortality seen in 37.5% of the cohort (n = 3), emphasizing the need for more effective treatments.

In addition to ineffectiveness of treatment, our cohort also demonstrated significant toxicity associated with current therapies. Foscarnet frequently causes nephrotoxicity and electrolyte imbalances and requires careful management with pre‐ and post‐hydration and monitoring of renal function and electrolytes [6, 10]. This side effect can be significant enough to require in‐hospital treatment and result in cessation of the drug, as was the case in 50% of the patients (n = 4) in our case series. Similar to foscarnet, cidofovir is also associated with nephrotoxicity and its administration requires IV hydration and probenecid [10]. One patient in our case series received cidofovir, and they experienced nephrotoxicity resulting in cessation of therapy. Due to the nature of HSCT and drugs utilized, this cohort is already vulnerable to renal injury, making any drug with nephrotoxicity as a side effect not an ideal choice for treatment. There is an unmet need for non‐nephrotoxic agents to manage resistant HSV infections in this cohort.

Immunocompromised hosts may also be at risk for temporal changes in resistance mutations, making treatment of acyclovir‐resistant HSV even more challenging. Case 5 in our series initially had a TK mutation identified and was treated appropriately with foscarnet; however, she continued to deteriorate despite treatment and developed severe ARDS due to HSV. In the context of her severe infection and failure to respond to treatment, resistance testing was re‐sent and demonstrated no TK mutation, but a new DNA polymerase mutation, which confers resistance to foscarnet [2]. As discussed, impaired ability of the host to clear HSV and thus allow ongoing viral replication, in the presence of antiviral treatment may increase selection pressure and temporal changes in resistance patterns. This case highlights the importance of reassessing resistance if there is no clinical response to antiviral treatment, even in cases where testing has previously been conducted.

While newer antivirals, such as helicase‐primase inhibitors (HPIs), have expanded the treatment options for acyclovir‐resistant HSV infections, access to these drugs can be challenging [2, 11]. HPIs are not affected by either TK or DNA polymerase mutations, thereby evading resistance mechanism for acyclovir and foscarnet. There is a case series in allogenic HSCT recipients for whom pritelivir has been used in acyclovir‐resistance HSV infections [12], and there is a current clinical trial being conducted to assess the efficacy and safety of pritelivir for treatment of acyclovir‐resistant mucocutaneous HSV infections in immunocompromised adult subjects (PRIOH‐1; NCT03073967). Pritelivir was provided on compassionate access for two cases in our series (Case 1and Case 5), but there was significant delay (> 20 days) between application, approval, and arrival of the drug and its duration of use was limited by the supplier. Access to HPIs rely upon compassionate access or enrolment on a clinical trial, meaning the practicality of accessing these treatments in a timely manner is not always feasible. This is even more challenging for pediatric patients, for whom there is no safety data or licensing.

There is limited evidence of novel therapies in the treatment of acyclovir‐resistant HSV infections. Adoptive T‐cell therapy involves the transfer of virus‐specific T cells from healthy individuals. There has been evidence for use of this therapy in other herpes viruses, including cytomegalovirus (CMV), Epstein–Barr virus (EBV), and herpesvirus 6 (HHV6) [13, 14], and feasibility of production of HSV‐1 specific T‐cell products has been shown [15, 16]. DLI was used in one of our patients (Case 5), it is hard to know whether this helped viral clearance as at the time of DLI being given the patient was critically unwell and proceeded to die shortly after. This treatment is also limited by donor availability, as we experienced.

Currently there exists no guidelines in pediatric patients for the investigation or management of acyclovir‐resistant HSV. We propose an algorithm for detecting and managing acyclovir‐resistant HSV in pediatric allogeneic HSCT recipients, as shown in Figure 1. HSV monitoring in blood should be performed if there is a clinical suspicion of HSV infection, and any associated lesions should be tested. HSV genotypic or phenotypic resistance testing should be considered in any patient with minimal clinical response to first‐line treatment (acyclovir) after 5–7 days. While awaiting results, patients should commence second‐line therapy with foscarnet [17]. With results of resistance testing, or if there is poor response to second‐line therapy after 5–7 days, consider third‐line therapy with either cidofovir, pritelivir, or amenamevir. Where possible, weaning of immunosuppression can help immune recovery and clearance of the virus. In refractory cases, adoptive T‐cell therapy and IvIg may play a role, though evidence to support their use is currently limited. Consider re‐testing for resistance if the patient is not responding to treatment.

FIGURE 1.

Proposed algorithm for investigating and managing acyclovir‐resistant HSV infections in pediatric allogenic HSCT recipients. DLI = donor lymphocyte infusion; GVHD = graft versus host disease; HSCT = hematopoietic stem cell transplant; HSV = herpes simplex virus. ¹Ref. [17].

Acyclovir‐resistant HSV is an uncommon, but emerging problem in pediatric recipients of allogeneic HSCT. Our case series highlights the severity of disease experienced by these patients, including significant morbidity and a high rate of mortality. Current treatment with foscarnet and cidofovir is limited by ineffective clearance of the virus and nephrotoxicity. Newer agents, such as HPIs, show promise, but access to these drugs is difficult, and is particularly challenging in pediatric patients, for whom there are no active clinical trials. Adoptive T‐cell therapy may play a role in supporting viral clearance. Improved awareness of this issue, early recognition, and early resistance testing, timely initiation of second‐line therapy, and weaning of immunosuppression are critical for disease control. There is a large unmet need for effective antiviral therapy in this cohort and collaborate pediatric registries will help in understanding the extent of this severe infection.

Supporting information

Appendix S1: Case summaries.