Norovirus Infection in Children with Acute Lymphoblastic Leukemia

Abstract

Norovirus infection in children on treatment for acute lymphoblastic leukemia can lead to severe morbidity due to chronic viral shedding, malabsorption, failure to thrive, and interruption of chemotherapy. We had four children with norovirus diarrhoea in eight years period in our pediatric oncology unit. Three children under two years of age had chronic noroviral shedding and persistent diarrhoea, probably due to poor adaptive immune responses. Two of those children didn’t respond to nitazoxanide and succumbed to the illness. The third patient who received nitazoxanide and favipiravir, is currently well on chemotherapy.

Diarrhoea is one of the common problems seen in oncology units. It could be because of chemotherapy or secondary due to various infections [1]. Norovirus is a non-enveloped single-stranded ribonucleic acid (RNA) virus belonging to the calciviridae family. It is responsible for most non-bacterial gastroenteritis in children. It is a major nosocomial threat due to its high infectivity (requiring as low as <100 particles to cause infection) and resistance to many alcohol-containing disinfectants (due to its non-enveloped nature) [2]. It’s infection in immunocompetent children can be self-limiting with asymptomatic viral shedding up to 2 - 3 weeks [3]. But in children with cancer on chemotherapy, hematopoietic stem cell transplants (HSCT), and immunodeficiency, it can lead to chronic infection with debilitating implications [4].Treatment for norovirus infection is usually supportive but various drugs with anti-viral action like nitazoxanide and favipiravir have been used off-label with varying success [5].

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Children younger than 2 years of age on treatment for ALL will have poor adaptive responses which makes them vulnerable to noroviral infection and its prolonged excretion, leading to a cascade of events including malabsorption, failure to thrive, and interruption of the chemotherapy leading to difficulty in management [2,5,6].

We did a retrospective analysis of children between 0 - 18 years with ALL treated at our center between December 2013 to July 2022, who had microbiologically proven norovirus infection. The epidemiological data, clinical status, type of ALL, chemotherapy protocol, and investigations of those children with norovirus infection were collected from hospital records and from an electronic patient database. Norovirus detection was done via the multiplex-polymerase chain reaction (PCR) method on stool samples. A structured protocol for testing stool from patients with diarrhoea for viral etiology was not in place and it was usually done on an individual case basis as per symptoms.

Four out of 161 children treated at our center for ALL had norovirus infection detected by multiplex-PCR. The test was outsourced to another lab where it was available (product detail: Allplex GI virus assay; lot number GI1332G02, Seegene Inc., Seoul, Korea) and a cycle threshold value of 36 was taken as the cut-off to report a viral nucleic acid detection. (Table 1).

Table1. Patient details, phase of treatment and outcome of children infected with norovirus.

Serial No.	Age at the time of diagnosis of Norovirus / Gender	Diagnosis	Phase of treatment at the time of diagnosis of Norovirus infection	Average Absolute lymphocyte count prior to the diagnosis (cells/mm3)	Noro virus species	Duration of Norovirus shedding	Treatment	Outcome
1	9 months / Female	Infantile ALL	Interfant 067 protocol – Induction B	441	GII	360 days (until death)	Nitazoxanide for 6 weeks	Failure to thrive, Relapsed ALL and death.
2	1 year 10 months / Male	Pre-B cell ALL	UK ALL 2003 Regimen B8 Consolidation	94	GII	165 days (until death)	Nitazoxanide for 8 weeks, intravenous immunoglobulins, total parenteral nutrition.	Failure to thrive and death
3	2-year-1 day / Male	Pre-B cell ALL	UK ALL 2003	882	GII	113 days	Nitazoxanide for 15 weeks and Favipiravir for 15 weeks	Currently on maintenance phase and clinically well
			Regimen A Induction			(stool negative for norovirus following 15 weeks of therapy)
4	5 years 2- months / Male	Pre-B cell ALL	UK ALL 2003	1,029	GII	14 days	No treatment	Recovered
			Regimen A Maintenance cycle 11

ALL, acute lymphoblastic leukemia; UK ALL 2003, United kingdom acute lymphoblastic leukemia 2003.

Child 1:

In this child, we performed the stool test for viruses when diarrhoea did not resolve by 7 days during induction B treatment for infantile ALL (Table1) [7]. She continued to have many bouts of diarrhoea (10 - 16 times/day) despite nitazoxanide 100mg twice-a-day therapy. She developed severe hypokalaemia, had weight loss, and failure to thrive. Due to her clinical condition intensive phase of chemotherapy was delayed by 30 days, and we also had to frequently interrupt her maintenance chemotherapy to allow her lymphocyte count to recover. Unfortunately, six months later her disease relapsed, and she passed away during the conditioning treatment for HSCT.

Child 2:

He developed diarrhoea towards the end of consolidation therapy [8]. A stool test was sent 4 weeks after the onset, during the interim maintenance block. He continued to have diarrhoea, persistent hypokalaemia, and failure to thrive. He was started on oral nitazoxanide 100mg twice daily, nasogastric tube feeding, and oral potassium supplementation. Despite this, diarrhoea persisted (7 - 8 bouts/day), and had a weight loss of 2.5 Kg (9 to 6.5 Kg). Lactose free diet and intravenous immunoglobulin therapy have been tried but showed no benefit in terms of frequency of unformed stools and weight loss. Norovirus excretion was persistent in stool even 60 days after starting treatment. Due to his poor clinical condition, there were intermittent delays in the chemotherapy schedule especially during the delayed intensification phase requiring dose modifications. Completion of the intense block of treatment was delayed by 37 days. He continued to have diarrhoea and weight loss. Four months after the onset of noroviral diarrhoea (first month of maintenance cycle 1) he had fever, pancytopenia and was in a poor clinical state requiring intensive care unit admission. We managed to start him on total parenteral nutrition (TPN) but he developed central line-associated gram-negative septicaemia and he was also positive for cytomegalovirus with copies of 125,000/dL and died.

Child 3:

This little boy developed diarrhoea and low-grade fever during the third week of remission induction. Owing to the neutropenic state, gram negative sepsis was our primary differential and was treated with empirical intravenous antibiotics. But as the diarrhoea and hypokalaemia persisted despite antibiotic therapy and as he was admitted into the hospital during patient 2's maintenance cycle 1, norovirus infection was suspected, and stool multiplex PCR done towards the end of induction detected it with a cycle threshold value of 16. In line with the treatment protocol developed at Great Ormond Street Hospital for Children, he was started on favipiravir 800 mg bis-in-die (BD) on day 1 followed by 200 mg BD in addition to oral nitazoxanide 100 mg BD and was nursed in isolation cubicle. He required oral potassium supplementation as well. His consolidation block was delayed by 30 days due to this. His stool was still positive for norovirus 30 days after treatment with a cycle threshold value of 22. He remained on this combination therapy with nitazoxanide and favipiravir throughout consolidation and interim maintenance. His adjunctive loperamide use and frequency of unformed stools were reduced, and his body weight remained stable. Fifteen weeks later stool was negative for norovirus (in the delayed intensification phase). We continued anti-viral therapy until the end of delayed intensification. He is currently doing well and in maintenance cycle 1 of chemotherapy.

Child 4:

He is a school-going boy and developed intermittent fever, abdominal pain and diarrhoea during maintenance cycle 11. Symptoms persisted for almost 7 days before testing for norovirus infection. His symptoms were self-limiting, and he recovered without any treatment.

Norovirus due to its high genetic diversity, is classified into 10 genogroups of which GI, GII, GIV, GVIII, and GIX are known to cause human infections, which are further divided into genotypes. GII.4 is the commonest genotype worldwide and was responsible for 6 major global pandemics. All our patients were infected with GII but genotyping couldn't be done [4]. Other diarrhoea causing viruses include rotavirus, adenovirus, astrovirus and sapovirus. Children 2, 3 and 4 were immunised with the rotavirus vaccine – children 2 and 3 received 3 doses of Rotavac (Bharat biotech international ltd, Medchal district, Telangana, India) and child 4 received 2 doses of Rotarix (GlaxoSmithKline Biologicals, Rixensart, Belgium). Though only 4 children were positive for norovirus, we believe this doesn't represent the true figure due to under-testing.

Norovirus is usually spread via direct feco-oral route, ingestion of aerosolized vomitus, fomites, and indirectly by contaminated surfaces and environment. Healthcare workers may also play a role in its nosocomial spread if stringent aseptic protocols aren't followed. Patients 2 and 3 developed norovirus infection around the same time in the hospital. Simon et al., reported an outbreak of norovirus in a span of 1 month in 11 patients in an oncology unit [9]. These patients and families spend a lot of social and recreational time together in the ward motivating and helping each other cope with the diagnosis and treatment. These factors along with common play areas, toilets, and breaches in aseptic protocols by healthcare staff and food handlers may contribute to outbreaks.

Host defence against norovirus depends on both innate and adaptive responses [10]. In a cohort of adults with common variable immunodeficiency, Brown et al. demonstrated a temporal relationship between low CD3+ and CD19+ cells and norovirus infection [5]. They also demonstrated in patients with chronic norovirus infection (CNI), reduced CD 27+ memory B – cells and increased PD-1⁺ T cells and CD27^- CXCR5⁺ B cells indicating immunological exhaustion. They also demonstrated villous atrophy in patients with CNI [5]. Similar to our series, children with hematological malignancies especially those under 2 years of age are most likely to develop CNI due to poor adaptive immune responses. This leads to prolonged viral shedding, persistent diarrhoea, villous atrophy, malabsorption, multiple and failure to thrive requiring a prolonged hospital stay. The morbidity and mortality are more in younger children with low absolute lymphocyte count irrespective of the treatment provided. They find it difficult to eradicate the virus due to a lack of memory B cells. Hence treating such children becomes a challenge especially with their underlying illness [9,11].

Child 4 who was 5 years old and had normal lymphocyte counts didn’t have CNI or a protracted course. Child 2 had a cytomegalovirus (CMV) co-infection which could have contributed to the morbidity but unfortunately, we cannot establish the time of infection or its causality to colitis as we tested for CMV only after he presented with cytopenias during the maintenance therapy.

Treatment of norovirus infection is supportive. Nitazoxanide is an effective drug against anaerobic bacteria, protozoa and some viruses. It has been reported to be efficacious against norovirus in some anecdotal reports and small studies in paediatric and adult patients with cancers and HSCTs [9,12]. Though its exact mechanism of action against norovirus is not known, it exerts its antiviral action by inhibiting protein synthesis and its active metabolite tizoxanide activated cellular antiviral response by stimulation of interferon stimulated genes in organoid models [12]. But we didn't find any benefit in our patients who were treated with nitazoxanide 100 mg BD. Patient 3 was treated with favipiravir in addition to nitazoxanide, and he remained stable throughout. Co-administering of antiviral drugs with different mechanisms of action may be required to clear a persistent RNA viral infection and will require further trials to validate this in a CNI. A case report by Ruis et al. reported favipiravir usage in an adult with primary immunodeficiency which reduced the symptom duration but had viral mutagenesis creating variants and drug-related side effects [13]. We used favipiravir for 15 weeks in our patient without any drug-related side effects. Favipiravir exerts its antiviral activity by inhibiting RNA dependent RNA-polymerase [14]. Adequate gut rest, medications, TPN and supportive therapy form the mainstay of therapy in immunocompromised hosts with prolonged symptoms. Providing TPN is a challenge in our setting where most of the patients are under treatment with financial aid by the government and non-government organisations and don't have an optimal nurse-to-patient ratio.

Though reducing immunosuppressive chemotherapy may seem a fair strategy given the poor overall clinical state of these children, this may be detrimental to curative outcomes of anti-leukemia therapy [12]. Patients 1, 2 and 3 required modifications in chemotherapy with an intent to allow the innate and adaptive responses to help in fighting norovirus infection. Prolonged treatment with Favipiravir and nitazoxanide may eradicate norovirus as happened in our patient 3, but we cannot make any concluding remarks without proper trials and further data.

Early suspicion of norovirus infection in children with leukemia is important to start them on anti-viral therapy, parenteral nutrition if required and optimization of electrolytes while making efforts to not compromise on the chemotherapy. The need to isolate these patients from other children with leukemia on treatment can't be emphasized enough.