Visceral leishmaniasis (VL) is a neglected protozoan disease caused by more than 20 Leishmania species. It is transmitted through the bite of sand flies from the genus Phlebotomus (Europe, Asia and Africa) and Lutzomyia (America) with an estimated incidence of 500,000 new cases per year, causing over 50,000 deaths every year worldwide.1,2
Leishmania infantum is responsible for most European cases of cutaneous and VL.2,3 Although dogs are the main reservoir, other mammals such as hares have been described.2,3 In Spain, VL is recognized as an endemic zoonosis with a mean annual incidence of 0.62 cases per 100,000 inhabitants.3,4
Children younger than 5 years of age and immunosuppressed adults are at highest risk. However, not all infections are associated with clinical manifestations and remain asymptomatic; therefore, the true incidence of Leishmania infections is likely underestimated.1,3–6
The diagnostic workup is not standardized and depends on the available infrastructure. Most common techniques include microscopic examination of tissue specimens including blood, bone marrow and spleen samples, antibody detection-based methods, such as enzyme-linked immunosorbent assay and indirect immunofluorescence, as well as polymerase chain reaction (PCR) (peripheral blood and/or bone marrow samples).7,8
Typically, patients present with a history of: fever without a source, hepato- and/or splenomegaly, adenopathy and weight loss. Cytopenia is common and hypergammaglobulinemia has been often reported.1,7,9,10
Secondary hemophagocytic lymphohistiocytosis (sHLH) has been described in the context of VL.9–13 Diagnostic criteria for HLH include clinical as well as laboratory parameters, many of which overlap with the typical VL manifestations.10,11,13 HLH-related symptoms are the result of cytotoxic T-cell and macrophage activation leading to an excessive proinflammatory cytokine production.14,15
Although liposomal amphotericin B (L-AmB) is the treatment of choice for pediatric VL in developed countries, there is no consensus regarding the best management defining details regarding the total accumulated dosage and administration regimens.7,16,17
In Southern Europe, data regarding the incidence, clinical manifestations, diagnostic procedures and management for pediatric VL are scarce.9
The aim of this study is to provide details about the disease burden and its characteristics, analyzing VL cases admitted to public hospitals in Andalusia (Southern Spain). In addition, we pursued to identify clinical and/or laboratory parameters that would allow detecting patients at risk developing complications such as sHLH.
MATERIALS AND METHODS
Study Setting and Patients
Pediatric infectious diseases specialists based at 10 Andalusian hospitals participated within the preestablished Grupo Andaluz de Infectología e Inmunopatología Pediátrica network. Pediatric patients (0–14 years old) with a diagnosis of VL during the study period (2004–2019) were retrospectively identified and data were extracted from the clinical charts and anonymized before analysis. This process was evaluated and approved by the regional Ethics Committee.
The diagnosis of VL was based on clinical manifestations (fever, hepatosplenomegaly and adenopathy), laboratory parameters and microbiologic testing including antibody detection tests, PCR on peripheral blood and/or bone marrow aspirate or the microscopic visualization of Leishmania parasites on blood or bone marrow samples.
sHLH case definition required the presence of greater than or equal to 5 of 8 criteria as stated in the hemophagocytic lymphohistiocytosis (HLH)-2004 protocol [fever, splenomegaly, bicytopenia, hypertriglyceridemia and/or hypofibrinogenemia, hemophagocytosis in bone marrow, spleen or lymph nodes, low or absent natural killer cell activity, hyperferritinemia and raised soluble CD25 (sCD25)].18 Laboratory parameters included full blood count, C-reactive protein (CRP), liver transaminases, fibrinogen, lactate dehydrogenase, ferritin, triglycerides and sCD25.14,18
Demographic characteristics, clinical manifestations, laboratory parameters, diagnostic tests, treatment protocols, responses and complications were extracted from the written charts or electronic data.
All variables were tested for normal distribution using the Kolmogorov-Smirnov test. Chi-square test or Fisher exact test was applied for the analysis of qualitative variables, whereas Student t test or Mann-Whitney U test was used in the case of between-group comparison of quantitative variables. Probability values were 2-tailed, and statistical significance was defined as P < 0.05. Analysis was performed using the IBM SPSS Statistics 25.0 for Windows (SPSS, Chicago, IL).
Description of the Overall Cohort
Over the study period, 127 pediatric patients were diagnosed with VL in the participating centers. The mean annual incidence was 18.2 cases per 100,000 pediatric hospitalizations. Although a median of 9 and 5 patients was diagnosed annually during the 2004–2008 and 2009–2014 periods, respectively, this number increased to 12 patients per year during 2015–2019 (see Figure, Supplemental Digital Content 1, http://links.lww.com/INF/E312).
Demographical and clinical data of the cohort are summarized in Table 1. Most children were ≤18 months old (61.4%). There was a predominance of female sex (57%) and 55% of patients were living in rural areas. Comorbidities were rare (6%) and included acute leukemia, nephropathies and pulmonary stenosis. Main symptoms and signs at the time of diagnosis were fever and splenomegaly (95% each), hepatomegaly (72%) and peripheral lymphadenopathy (21%). The median duration of fever and length of hospitalization were 14 (interquartile range, 10–20) and 10 days (interquartile range, 7.5–15 days), respectively.
TABLE 1. -
Demographical and Clinical Data of 127 Patients
|Clinical Findings in the Overall Cohort
||Number of Patients (%)
|Median age (IQR), mo
|Living in rural area
| Median duration in days of fever (IQR)
| Median length of hospitalization in days (IQR)
| L-AmB iv
| Relapses (%)
| Adverse events (%)
| Meglumine antimoniate IM
| Relapses (%)
| Adverse events (%)
| L-AmB iv + miltefosine
| Relapses (%)
| Adverse events (%)
IM indicates intramuscular; IQR, interquartile range; iv, intravenous.
Direct microscopy of bone marrow aspirates showed Leishmania amastigotes in 39 patients, although the results of this method were not available in all patients. Serologic tests revealed positive results for IgG (87%, 96/110) and/or IgM (70%, 33/47). For 80 patients, peripheral blood or bone marrow samples were tested by PCR, being positive in 70% of cases.
HLH-defining laboratory findings of the overall cohort are summarized in Table 2. About 29.1% of the patients fulfilled the diagnostic HLH criteria, all of which were deemed to be sHLH in the absence of a suggestive personal or family history. Details regarding the presence of HLH-related genetic variants are not available; however, none of the patients has developed HLH during the clinical follow-up. In our cohort, cytopenia was 1 of the most characteristic features. Pancytopenia was observed in 49% of the patients. Anemia was most common (80%), followed by thrombocytopenia (68%) and neutropenia (63%). Inflammatory markers (eg, CRP and ferritin) were raised in about half of the patients.
TABLE 2. -
Laboratory Findings in the Overall Cohort
|Total Number of Patients
|Hemoglobin at admission (g/dL), mean ± sd
||8.15 ± 1.8
|Hemoglobin minimum (g/dL), mean ± sd
||7.08 ± 1.54
| Anemia (Hb < 9 g/dL), n (%)
|Leukocytes at admission (cel/μL), median (IQR)
|Leukocytes minimum (cel/μL), median (IQR)
| Neutrophils at admission (cel/μL), median (IQR)
| Neutrophils minimum (cel/μL), median (IQR)
| Neutropenia (<1000 cel/μL), n (%)
|Platelets at admission (cel/μL), median (IQR)
|Platelets minimum (cel/μL), median (IQR)
| Thrombocytopenia (<100,000 cel/μL), n (%)
|Pancytopenia, n (%)
|CRP highest value (mg/L), median (IQR)
| CRP > 50 mg/L, n (%)
|TGs (mg/dL), median (IQR)
| Hypertriglyceridemia (TG > 250 mg/dL), n (%)
|Ferritin (mg/dL), median (IQR)
| Hyperferritinemia (>500 mg/dL), n (%)
|Soluble CD25 (soluble IL-2 receptor), median (IQR), IU/mL
| Elevated sCD25 (≥2400 IU/mL) (%)
Hb indicates hemoglobin; IQR, interquartile range; TG, triglyceride.
Treatment and Outcome of the Overall Cohort
L-AmB was the most frequently prescribed drug (97%). Total treatment dosing ranged from 18 to 21 mg/kg. Two treatment regimens were most commonly used: 3 mg/kg/d intravenous days 1–5 and 10th day (n = 63, 50%) or 3 mg/kg/d intravenous days 1–5, and 14th and 21st days (n = 23, 18%). Intramuscular meglumine antimoniate was only used in the first year of the study period (n = 3). Detailed information regarding the applied treatment regimens was not available for 37 patients.
One patient in the context of a renal transplant with immunosuppression (tacrolimus and mycophenolate) received combination therapy with L-AmB in association with miltefosine. As he also developed clinical and laboratory signs compatible with sHLH, corticosteroids were added. He recovered without relapse.
Only 4 patients (4/127, 3%) relapsed after L-AmB treatment and resolved after a second course of L-AmB. These children showed more pronounced pancytopenia (mean hemoglobin, leucocyte and platelet count 5.97 g/dL, 3302 cel/μL and 33,500 cel/μL, respectively) and marked elevated CRP levels (mean values 147 mg/L) during their admission.
Seven cases (5.5%) required pediatric intensive care unit (PICU) admission (6/7 with sHLH) and 1 of these patients died in the context of a multiorgan failure.
Comparison of VL Patients With or Without sHLH
Demographic and clinical characteristics, laboratory results and treatment outcomes for these 2 patient groups are summarized in Table 3.
TABLE 3. -
Demographic, Clinical Characteristics, Laboratory Parameters and Treatment Outcomes in Patients With (n = 37) or Without sHLH (n = 90)
|Clinical and Laboratory Data
||Children With sHLH
||Children Without sHLH
|Age at diagnosis, mo, median (IQR)
|Days of fever before admission, median (IQR)
|Days of admission, median (IQR)
|Hb at admission (g/dL), median (IQR)
|Leukocytes at admission (cel/μL), median (IQR)
|Neutrophils at admission (cel/μL), median (IQR)
||875 (460 - 1470)
|Lymphocytes at admission (cel/μL), median (IQR)
|Platelets at admission (cel/μL), median (IQR)
|Platelets <83,500/μL (%)
|Monocytes at admission (cel/μL), median (IQR)
|CRP highest value (mg/L), median (IQR)
|Ferritin maximum, μg/L, median (IQR)
|Hypertriglyceridemia (TG > 250 mg/dL) (%)
|Platelet transfusion during admission, n (%)
|Red blood cell transfusion during admission, n (%)
|PICU admission, n (%)
|Alive and cured, n (%)
IQR indicates interquartile range; TG, triglyceride.
Gender, age, days of fever before admission or living in rural areas did not differ between the 2 categories. However, 62% (5/8) of children with comorbidity developed sHLH.
Patients with sHLH (29.1%, 37/127) were more likely to present pancytopenia (70.3% vs. 40%, P = 0.007). Thrombocytopenia at admission was common in both groups but more pronounced in the sHLH group (median = 72,000 vs. 98,000 cells/uL, P = 0.002). Interestingly, absolute monocyte counts, a feature not included in the HLH criteria, were significantly lower in the sHLH group (median = 200 vs. 360 cells/uL, P = 0.011). The analysis of inflammatory markers revealed that hyperferritinemia was not only more common (86 vs. 35%, P = 0.0001) but also higher in the sHLH group (median = 2739 vs. 534 μg/L, P = 0.001). Similar observations were made for the CRP levels (median = 88.5 vs. 53 mg/L, P = 0.031). The determination of sCD25 levels was available for 11 sHLH patients and elevated in all of them (median = 2280.3 IU/mL). Finally, hypertriglyceridemia (item included in the HLH criteria) was found to be more common in the sHLH group (73 vs. 23.6 %, P = 0.0001).
For both groups, L-AmB was the most commonly prescribed treatment. No differences were observed in terms of total dosage although detailed data were not available for each patient. Thirteen of the 37 patients (35%) diagnosed with sHLH-received adjuvant immunomodulatory therapy. Monotherapy with corticosteroids (n = 8) or in combination with other immunosuppressive drugs including cyclosporine, intravenous polyvalent immunoglobulins and etoposide (n = 5) were the therapies used. These regimens were administered in addition to the specific leishmaniasis treatment and were not associated with disease relapses.
Clinical Course and Outcome
The clinical course of sHLH was associated with prolonged hospital admissions (14 vs. 10 days, P = 0.001) and an increased need for platelet (P = 0.006) and red blood cell transfusions (P < 0.0001). Furthermore, PICU admission was more likely in patients with sHLH (P = 0.0007).
We describe epidemiologic and clinical features, laboratory parameters, treatment regimens, complications and the overall outcome in pediatric patients diagnosed in Southern Spain during a 15-year period (2004–2019). To the best of our knowledge, this is the largest pediatric VL cohort from Europe to date.9,13,19–21
As patient’s recruitment was performed systematically, using the Andalusian network of pediatric infectious diseases (Grupo Andaluz de Infectología e Inmunopatología Pediátrica), our data likely represent most recorded pediatric VL cases during the study period. The VL incidence in our area was 18.2 cases per 100,000 pediatric hospitalizations, being inferior when compared with a recently performed study in a single pediatric children hospital in Greece (51/100,000 hospitalizations).9 The mean annual incidence in our cohort was 8 cases per year with a high variability. Interestingly, between 2015 and 2019, the annual number increased to 12 patients per year. Increased clinical awareness, data availability (eg, electronically charts) and the more extended use and availability of diagnostic tools might have contributed to the observed increase of diagnosed patients.
Children between 8 and 24 months of age (median = 14 months) showed the highest incidence of VL. Although the age of our patients was lower than other studies, the increased VL risk in early infancy, likely resulting from an immaturity of the developing immune system, has also been highlighted in previous studies performed in Southern Europe.9,13,19–23
In concordance with other series, in our cohort, the classical symptoms such as fever and splenomegaly were the most common clinical findings (95%), followed by hepatomegaly (72%).9,10,19,20,24 As previously reported, anemia (80%) and thrombocytopenia (68%) were the most frequent hematologic abnormalities at presentation, pancytopenia was found in 49% and CRP levels were elevated in 52% of the patients.9,10,19,20,24
In this retrospective study, the diagnostic process was highly heterogeneous. Most of our cases were identified by clinical manifestations in combination with either visualization of the pathogen, serology or PCR. The high variability of diagnostic testing is likely because of the extended study period, local preferences and availability of diagnostic methods such as PCR. Unfortunately, detailed information regarding the diagnostic workup was not available in many patients. This, in addition to in-house implementation of molecular methods in the different centers, may explain the relatively low diagnostic yield of methods such as PCR in blood or bone marrow samples.25 To optimize the diagnostic process, our group is currently working on a specific guideline for pediatric patients under investigation for Leishmania infections for our area.
L-AmB is considered the first-line treatment of VL in Europe.1,12,17,26 In our cohort, 97% of patients received intravenous L-AmB in monotherapy following 2 main regimes (total dose = 18–21 mg/kg), with excellent outcomes. Treatment relapses (3%) as well as severe adverse events (4%) were uncommon and comparable with previous studies.9,13,17,19,21,27 Mild adverse effects such as low-grade fever related to L-AmB administration were not registered in our study.
The L-AmB treatment regimens prescribed in our cohort are in line with the most widely available recommendations.9,12,17,26,27 Interestingly, Krepis et al17 reported high cure rates (96%) and only mild adverse events using a short-course regimen (10 mg/kg/24 h for 2 days) in a retrospective study. Shorter intensive treatment regimens with L-AmB appear not to be inferior as the observed frequency of relapses was similarly to our study (2.3% vs. 3%).9,17 However, the effectiveness and limitations of short-course treatment strategies remain to be determined in prospective randomized studies. Notably, the median length of hospitalization was 10 days in our cohort, being shorter than patients treated with antimonial drugs in Brazil (29 days).10 Not surprisingly, meglumine antimoniate was only used in 3 patients at the beginning of our study period, as antimonials are no longer considered first-line therapy in Europe.1,17,27
Treatment responses and full clinical recovery were observed in all but 1 patient after receiving specific VL therapy.
sHLH in the context of VL has been previously described.9–13,21 In our study, 29% of our patients met the established HLH criteria. These numbers are comparable with those described by other groups such as Daher et al10 (27.5%), Clavijo et al21 (32%) and Blázquez-Gamero et al13 (41%), but are higher than those observed in the largest Greek cohorts (9.3%).9 Of note, Spain was also reported to represent the most common area of imported VL infection (48%) in a pediatric HLH cohort in 2014.12
The overlap of clinical as well as laboratory manifestations between VL and HLH often results in diagnostic dilemmas. These findings further justify to include VL in the differential diagnosis of HLH and that the systematic evaluation for a potential Leishmania infection should be considered as part of the routine workup of patients with suspected HLH aiming to avoid unnecessary and potentially harmful high-dose immunosuppression (eg, HLH-2004 treatment protocol). In fact, preemptive L-AmB therapy might be advisable when specific HLH workup is not possible or delayed, given the low risk of serious adverse advents and the often-prompt clinical response. The consultation with an infectious disease specialist in patients under investigation for HLH is recommended.
As expected, significant differences were found when comparing parameters included in the most commonly used HLH-2004 criteria such as platelet count, ferritin and triglyceride levels in patients with or without HLH.13,21
Furthermore, we could show that alterations in these markers were significantly more pronounced in patients with sHLH. In addition, we describe for the first time new variables not included in the HLH criteria such as a low monocyte count at admission. Monocytes are known to play an important role in the setting of Leishmania infection.28,29 Proinflammatory cytokine environments in the affected tissues are necessary to control adequately the infection and are associated with an accelerated monocytopoiesis resulting in peripheral blood monocytosis during the acute phase.28 Subsequently, a transient monocytopenia may occur in an attempt to control the parasite as they migrate from the peripheral blood stream to other infected tissues, differentiating into tissue macrophages and thereby causing “transient monocytopenia” on the peripheral blood smear.28,29 Bone marrow dysfunction secondary to a chronic Leishmania infection has been described and this mechanism may also contribute to the here observed monocytopenia.30 Interestingly, monocytopenia has been shown to correlate with an increased parasite load in peripheral blood in animal models.31 Whether the here-observed monocytopenia is caused by the sequestration of circulating monocytes to the infected tissues as a response to a high parasite inoculation and/or if it is rather because of an impaired overall bone marrow dysfunction secondary to a chronic Leishmania infection will need future studies. Interestingly, no such differences were detected for the rest of the white blood cell compartment.
Furthermore, the proinflammatory markers CRP, sCD25 and ferritin levels were significantly higher in sHLH patients during the clinical course. Whether these variables alone or in combination may be used to predict the risk of sHLH development or other relevant complications remains to be determined.
sHLH in the context of VL is a serious complication and reflected by longer hospital stays, higher rates of red blood cell and platelet transfusions as well as PICU admissions.
In our cohort of 37 patients diagnosed with VL and HLH, 24 (65%) received L-AmB monotherapy and 13 (35%) additional immunosuppressive therapy; all patients showed a complete clinical and laboratory recovery with no complications or treatment relapses. These results are comparable with previous reports.9,12,13,21 Whether some selected patients may benefit from adjuvant low-dose immunosuppression or if exclusive antiparasitic treatment with L-AmB is sufficient to control and treat sHLH as well remains to be clarified.
The main limitations of this study are related to its retrospective design. Clinical and laboratory data were not available for some patients. Furthermore, diagnostic testing and therapy varied between the participating centers and also over the time of the study period. Firm conclusions regarding the performance of the employed diagnostic methods or the efficacy of treatment strategies are therefore limited. Finally, an immunology workup aimed to identify primary HLH or other inborn errors of immunity was not performed.
In summary, this large retrospective study on children suffering from VL highlights the importance of correctly diagnosing and managing this disseminated infection being commonly associated with sHLH and predominantly presenting before 2 years old. We report for the first time raised CRP levels and monocytopenia as additional risk factors for the development of sHLH in the context of VL. Future challenges include elaborating a diagnostic and treatment consensus on medications, doses and duration as well as the identification of laboratory markers allowing to distinguish VL from VL with HLH, the latter being important to initiate additional supportive clinical care and immunomodulatory therapy.
The authors thank to all members of Grupo Andaluz de Infectología e Inmunopatología Pediátrica (GAIIP). The authors also thank Eloisa Rubio Pérez from the Statistics and Research Methodology Unit of the Fundación Pública Andaluza para la Gestión de la Investigación en Salud de Sevilla (FISEVI) for her statistical advice on this study.
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