Combined immunodeficiencies (CIDs) comprise a heterogeneous group of disorders with impaired development, function, or both, of T lymphocytes associated with a defective antibody response.1 In the most severe forms, also known as severe combined immunodeficiencies (SCID), there is a virtual lack of functional peripheral T cells and children present early in life with recurrent and severe infections that are fatal without treatment.1,2
Hematopoietic stem cell transplantation (HSCT) is the standard treatment, potentially inducing long-lasting reconstitution of immunity, although some patients could benefit from enzyme replacement therapy (ERT) or gene therapy.3,4 Survival after HSCT has been strongly related to patient’s age and clinical situation at the time of transplantation.5 Systemic, respiratory and gastrointestinal infections lead to malnutrition, hepatic and respiratory impairment that worsen prognosis.5 Early diagnosis is therefore critical, as it enables us to start searching for a donor, to establish nutritional support, antibiotic prophylaxis, immunoglobulin replacement therapy and other preventive measures to avoid infections, improving survival.
The aim of our study is to describe clinical features before starting treatment (HSCT or ERT) in our cohort of children with CID treated in a National Reference Unit for primary immunodeficiencies (PIDs).
PATIENTS AND METHODS
We conducted a descriptive and retrospective study of hospital records of children with CIDs treated in our Hospital during a 20-year period (1995–2014), analyzing their clinical outcome before HSCT or ERT with pegylated bovine adenosine deaminase. HSCT remains the mainstay of treatment in our Hospital, although ERT is an alternative therapeutic option in adenosine deaminase deficiency when an HLA-matched donor is not available.
We included children with a definitive CID diagnosis according to the updated classification of the International Union of Immunological Societies,6 and children with probable SCID according to the European Society for Immunodeficencies criteria.7 CIDs with associated or syndromic features (such as Wiskott-Aldrich syndrome, DiGeorge syndrome, ataxia–telangiectasia and cartilage hair hypoplasia) were excluded,6 as well as noncombined PIDs. Standard diagnostic work-up included immunological evaluation and gene sequencing in all patients.
A database was specifically designed for the study, including the following variables: age at clinical presentation, at diagnosis and at transplantation/ERT, baseline and nadir lymphocyte count, immunoglobulin levels at diagnosis, type of CID, risk factors including family history of CID, infant deaths or consanguinity, clinical status at diagnosis and clinical outcome and management before transplantation/ERT.
Periodic virological surveillance of asymptomatic patients was performed in all cases before HSCT since 2010, including the collection of nasopharyngeal aspirates (NPA), cytomegalovirus (CMV) urine culture and CMV antigenemia or blood polymerase chain reaction (PCR). Before 2010, these tests, bronchoalveolar lavage (BAL) or PCR for DNA viruses were only performed when clinically indicated. NPA specimens were processed using rapid tests for respiratory syncytial virus (RSV) and influenza A and B viruses, and viral culture for adenovirus, parainfluenza, influenza and enterovirus. If the rapid influenza antigen test was negative and the child presented suggestive symptoms during epidemic periods, a PCR was also performed.
BAL samples underwent bacterial, mycobacterial, fungal and viral cultures, as well as PCR for DNA viruses and cytologic evaluation for the presence of Pneumocystis jirovecii. Since 2012, a PCR to detect 16 respiratory viruses was also performed in all BAL samples (CLART PneumoVir, GENOMICA, Madrid, Spain).
Categorical variables were described with absolute and relative frequencies. Continuous variables were described with median and interquartile range (IQR). Data analysis was performed using SPSS statistical software, version 18.0 (SPSS Inc., Chicago, IL).
Thirty-one children with CID were treated in our Hospital during the study period, 23 boys (74%) and 8 girls (25%). Twenty-five children (81%) had been referred to our Unit from other hospitals to confirm diagnosis and/or start treatment. Twenty-six children underwent HSCT, 2 died before HSCT, 2 started ERT with pegylated bovine adenosine deaminase at the age of 2 and 13 months, respectively, and 1 patient is still waiting for HSCT. Risk factors for CID were present in 11 of 31 cases (35%), including family history of CID (5/31), infant deaths (4/31) or consanguinity (3/31). Three children were studied because of family history of CID and were asymptomatic at diagnosis. The rest of patients were diagnosed after a varying duration of symptoms. Median age at clinical onset, diagnosis and HSCT/ERT was 3.3 months (IQR: 2.1–5.1 months), 5.6 months (IQR: 4.1–9.9 months) and 8.1 months (IQR: 6.7–14.5 months), respectively. Nine patients (29%) required intensive care unit admission at clinical presentation. Twenty-nine patients (93%) required hospital admission between diagnosis and HSCT/ERT due to infectious diseases, respiratory failure or nutritional support necessity.
At the time of admission to our unit, 58% of patients had bronchiolitis-like illness (18/31) and 68% (21/31) presented abnormal lung auscultation. Before HSCT/ERT, respiratory infections were documented in 80% of cases (25/31), detecting RSV antigen in 8 NPA samples (32%; Table 1). Fifteen BAL were performed before HSCT/ERT in 13 of the 31 children (42%), identifying pathogens in 11 of them (73%, Table 1).
Median birth weight percentile of our patients was 50 (IQR: 19–71). Nevertheless, median weight percentile at admission in our unit was 7 (IQR: 0–29), and 72% of cases (18/25) presented a Waterlow Index below 90%.
At admission, 14 patients reported chronic gastroenteritis (45%), and before HSCT/ERT, 84% presented diarrhea (26/31). Pathogens were identified in 19 stool samples of 18 patients with persistent gastrointestinal symptoms (Table 1). Twenty-two children (71%) received nutritional support before HSCT/ERT because of failure to thrive, chronic diarrhea or feeding difficulties (15 enteral feeding through nasogastric tube and 7 parenteral nutrition). Physical examination at admission revealed hepatosplenomegaly in 35% of cases (11/31), and 13 children (42%) had raised serum transaminases at diagnosis.
Fifty-five percent of patients (17/31) had skin manifestations: 9 moderate/severe atopic dermatitis (29%), 4 cutaneous candidiasis (13%), 3 mateno-fetal graft versus host disease (GvHD) confirmed by skin biopsy (10%), 1 Trichophyton dermatophytosis (3%) and 1 disseminated molluscum contagiosum (3%). Recurrent oral thrush was observed in 42% of cases before diagnosis (13/31).
Between hospital admission and HSCT/ERT, 15 sepsis episodes were diagnosed in 13 out of 31 patients (42%, Table 1). In 7 children (23%), viral systemic infection was confirmed by PCR: 4 CMV, 1 Epstein–Barr virus (EBV), 1 herpes 6 virus and 1 enterovirus. All children with CMV or EBV infections received intravenous ganciclovir until they were asymptomatic and with at least 2 consecutive negative blood viral load results. One child with adenovirus infection received intravenous cidofovir until he died. Specific RSV treatment was given to 2 out of 8 infected children: intravenous palivizumab in 1 case, and inhaled ribavirin plus intravenous palivizumab in the other. Influenza infection was confirmed in 2 patients, one of whom received oral oseltamivir.
The median baseline and nadir lymphocyte count was 2374/mm3 (IQR: 890–3982/mm3) and 680/mm3 (IQR: 243–2240/mm3), respectively. All patients presented a lymphocyte count lower than 4000/mm3 before HSCT/ERT. The median IgG levels at diagnosis was 305 mg/dL (IQR: 64.4–615 mg/dL). Patients with SCID and no maternal engraftment presented very low number of T cells compared with normal age (CD3 T cells < 500/mL in all cases). No detectable lymphocyte proliferative response to mitogens or recall antigens (candidin or tetanus toxoid) was detected in any case of SCID. Patients with MHC class II deficiency presented CD4 lymphopenia, and reduced proliferative response to recall antigens, but normal response to phytohemagglutinin. In these cases, less than 5% of B cells or monocytes expressed superficial HLA-DR by flow cytometry assays. The patient with CD40 ligand deficiency presented normal counts of T and B cells, but no expression of superficial CD40L was detected on CD4 cells by flow cytometry. Diagnosis was confirmed by genetic studies, which showed a pathogenic mutation in the CD40LG gene.
Types of CID diagnosed in our patients are described in Table 2. A causative genetic mutation was identified in 25 of the 31 cases included (81%), and in 20 of the 26 SCID cases (77%). Before HSCT/ERT, all patients received immunoglobulin replacement therapy and P. jirovecii prophylaxis, 30 children with cotrimoxazole and 1 child with dapsone due to hypersensitivity to cotrimoxazole. Nineteen patients who had parenteral nutrition, prolonged use of broad-spectrum antibiotics, central venous lines or mucocutaneous candidiasis received antifungal prophylaxis (61%). One patient died due to pulmonary aspergillosis, although he was under antifungal prophylaxis. Previously to Aspergillus infection, he was critically ill, severely malnourished, requiring total parenteral nutrition, broad-spectrum antibiotics and mechanical ventilation. No other cases of invasive fungal infection were observed in our series.
The mortality rate was 35% (11/31). Two patients died before HSCT and 9 after it. One of the 3 children studied due to family history died at the age of 9 months. Although he was promptly diagnosed, he underwent HSCT at 7 months old because an HLA-matched unrelated donor was not found before. The other 2 patients studied at birth due to family history successfully underwent HSCT/ERT before 3 months old. The 2 patients treated with ERT are currently 11 and 16 years old and remain asymptomatic. Causes of death are listed in Table 3.
CIDs are a heterogeneous group of disorders of diverse genetic cause characterized by profound deficiencies of T and B cell function.1 SCID is a pediatric emergency,8,9 and those patients who do not accomplish immune reconstitution usually die from infection within the first 2 years of life. Some CIDs not considered classical SCIDs, such as MHC type II deficiency, can also have severe infections and clinical findings of typical SCID.10 For this reason, we decided to include them in the study.
Recent studies have shown that before being diagnosed of their immunodeficiency, 70%–90% of patients present one or more infections,10,11 48% persistent bronchiolitis-like illness and 14%–23% develop pulmonary dysfunction with oxygen requirement before HSCT.2 All these conditions worsen these patients’ prognosis. Respiratory and DNA viruses (32%–36%), bacteria (22%–43%) and P. jirovecii (16%–25%) are the most common documented infections, whereas other fungal infections are less frequent (2%–16%) and mycobacterial disease is exceptional (1%–2%).2,10,11 The outcome varies depending on the causative microorganism. In our cohort, 72% of children who died had a Gram-negative bacterial sepsis or a viral infection. Respiratory and DNA viruses are likely to remain active once patients have been infected, which significantly worsens HSCT outcomes.11,12 Functional T-cell immunity is required to clear viruses, and most deaths after HSCT are caused by viral systemic infections or pulmonary complications of viral respiratory infections.11,13 Thirty-two percent of children in our series were infected by RSV before HSCT/ERT. Prior studies show that patients with paramyxovirus infection usually develop severe pneumonitis that worsens after transplantation, with a survival rate below 50%.12
Prevention and prompt treatment of viral infections is therefore crucial in the management of CID patients. All children should receive strict reverse barrier nursing under laminar flow isolation during admission.9,12 Blood products must be irradiated and CMV-negative and breastfeeding should be discouraged until mother’s CMV status is known.9 Palivizumab prophylaxis is recommended for prevention of RSV infection,14 and all household members older than 6 months should receive influenza vaccine.15 Some authors also recommend weekly virological surveillance of asymptomatic children,9,12 as early detection can improve prognosis.12,16 Bacteremia also predicts poor outcome after HSCT in children with SCID,5 although bacterial infections are more likely to be resolved with appropriate antimicrobial treatment.11,17
Early diagnosis of respiratory infections is often difficult, as clinical and radiological findings lack sensitivity and specificity, and performance of BAL is usually necessary to identify the causative microorganism.18,19 Specifically, the use of PCR in BAL samples enables early etiologic diagnosis, helps to direct antimicrobial treatment and leads to a clinical improvement in 60% of cases.12,18,19 However, although up to 80% of our patients had respiratory infections, BAL was only performed in 42% of cases, probably due to fear of worsening respiratory status in patients who already had respiratory impairment. Nevertheless, most authors have reported a low incidence of complications, even in symptomatic patients.18,19
A high proportion of children in our series presented failure to thrive secondary to gastrointestinal infections or feeding difficulties due to respiratory infections. Marron et al20 demonstrated that increased resting energy expenditure is common in these patients, even in those asymptomatic, and may contribute to the development of failure to thrive. Since malnutrition is also associated with poor transplantation outcome,5 these patients require intensive nutrition support before HSCT.9,20 Pre-existing liver impairment is also associated with a worse outcome,5 and 35% of children in our cohort presented hepatosplenomegaly and 42% raised serum transaminases at diagnosis. In addition to severe infections and failure to thrive, infants may also present with different skin manifestations, ranging from fungal, viral or bacterial infections to generalized eczema.21 Materno-fetal GvHD secondary to transplacentally transferred maternal T lymphocytes is the cause of eczema in a small percentage of patients. This transfer is common,2,11 but only in 9% of cases associates GvHD.2 In our cohort, 10% of cases developed cutaneous GvHD before transplantation.
Early diagnosis of CID is associated with better HSCT outcomes.8,11 Infants who undergo HSCT before 3.5 months old have a survival rate higher than 90%.11 In addition, successful engraftment after HSCT is more likely in young children.22,23 However, median age of our patients at transplantation was 8.1 months. Similarly, Pai et al11 reported that only 11% of their SCID patients received an early HSCT. There are some key clues to immunodeficiency in routine diagnostic tests, including persistent lymphopenia, particularly in the first months of life, and an absent thymic shadow on chest radiograph.8 All our patients had lymphocyte counts below 4000/mm3 before HSCT/ERT. In addition, other indicators of PID such as parental consanguinity, history of early deaths in childhood or family history of SCID are frequently present as we have observed.10,21 However, although all our patients had lymphopenia and most of them had suffered relevant infections, or had a positive family history, these factors were not early identified. Other authors have also reported that diagnosis of SCID is often delayed due to failure to recognize these early warning signs.8 A single episode of severe infection early in life, especially when associated with lymphopenia, should be enough to consider CID as a possible underlying cause,21 and any lymphocyte count lower than 2500/mm3 must be rechecked.8–10 A persistently low count requires further investigation in a specialized center for PIDs.9,24 However, some children with CID may have near normal absolute lymphocyte counts with no positive family history,9,10,21 and they are only diagnosed after experiencing cumulative and serious infections.8,22 In the past years, the identification of infants with SCID using DNA isolated from dried blood collected for newborn screening is possible, by measuring T-cell receptor excision circles, a biomarker for T lymphopoiesis.25–27
Since 2008, a number of US states have successfully adopted newborn screening of SCID using the T-cell receptor excision circles assay. The high survival of infants diagnosed as newborns demonstrates the benefit of universal screening.9,25,26 In Europe, De Pagter et al28 observed that, in the absence of neonatal screening, mortality due to fulminant infections was not prevented in a considerable proportion of cases, despite early diagnosis was performed. For this reason, we believe that newborn screening is the only way to improve the outcome of these children, by diagnosing them before the onset of symptoms and the development of infectious complications.28 However, although there is strong evidence that SCID fulfills the internationally established criteria for newborn screening, it has not been implemented in Europe yet.27
Until it is available in our country, early diagnosis is crucial to prevent severe infections and organ impairment. Infants should ideally undergo HSCT at 3.5 months or younger, but older patients have similar survival rates when there are no active infections at the time of transplantation.11P. jirovecii prophylaxis and immunoglobulin replacement therapy are recommended in all patients.9 Almost two-thirds of our patients received antifungal prophylaxis, although it has not been specifically evaluated for SCID patients and it depends on local guidelines and clinical circumstances.9,24
One of our patients died despite having been diagnosed as a newborn, as HSCT could not be performed in a timely manner due to the lack of an HLA-matched unrelated donor. Although allogeneic HSCT from HLA-identical donors is the best option, it is available for only about 25% of children.29 However, survival rates among young infants at the time of transplantation are very high regardless of donor type, another important reason to recommend neonatal screening.
The main limitation of our study is its retrospective design. We could not accurately estimate the real infection rate in this population, because infection surveillance was not performed identically in all patients. However, our findings suggest that viruses are the main source of infection with important implications in clinical outcome. Probably, infection rates would have been even higher if all children had undergone additional examinations like routine BAL or systematic weekly virological surveillance. On the other hand, the present study shows the characteristics and outcome of children with CIDs treated in a specialized pediatric hospital, in the absence of neonatal screening. Our results highlight the great importance of the implementation of SCID newborn screening in our country and the early referral of these patients to a specialized PID center, to start treatment.
The authors would like to acknowledge Dr. Roberto Hernandez for his encouragement. This study was carried out thanks to his constant support.
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