Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder characterized by an uncontrolled activation of T lymphocytes, natural killer cells and macrophages, leading to secretion of high levels of proinflammatory cytokines.1–3 This syndrome can be caused by genetic defects (familiar HLH and HLH associated with immune deficiency syndromes) or associated with some underlying conditions among which infections are the most frequent.1,2,4 Diagnosis of HLH is based on genetic, clinical and laboratory criteria,1 but clinical manifestations usually overlap between the 2 forms of HLH.
Visceral leishmaniasis (VL) is a systemic disease caused by the protozoan pathogen Leishmania infantum or Leishmania donovani. In an HLH national database in Germany, Leishmania was the second infectious trigger after Epstein–Barr virus (EBV).5 The prevalence of HLH associated with VL is unknown but is thought to be an uncommon complication.6–12 The aims of this study were (1) to determine how many children with VL met the HLH criteria during a community outbreak of L. infantum infection in the Southwest area of Madrid and (2) to evaluate the outcome of VL-associated HLH to specific anti-Leishmania treatment.
Setting and Study Population
A multicenter prospective study was conducted in 7 hospitals in Madrid between January 2010 and December 2011. All children younger than 16 years with confirmed diagnosis of VL were included. Informed consent was obtained from parents or legal guardians. This study was approved by the Institutional Review Board of 12 de Octubre University Hospital.
Diagnosis of VL was made on the basis of clinical manifestations and detection of Leishmania in blood and/or bone marrow (BM) aspirates, with at least 1 of the following methods: direct visualization, culture in appropriate media and/or Leishmania nested polymerase chain reaction (LnPCR). Diagnostic criteria for HLH were those proposed in 2004 by the Hemophagocytic Lymphohistiocytosis Study Group.1
In every patient with clinically compatible VL, complete blood cell count, triglycerides, plasma ferritin and fibrinogen were determined within the first 96 hours after admission. Soluble interleukin-2 receptor (sCD25) in plasma was measured by an enzyme-linked immunosorbent assay (R&D, Abingdon, UK) within the first 96 hours after admission and was available only in 1 center. BM culture was performed inoculating 100 μL BM aspirate in Novy–MacNeal–Nicolle medium. Cultures were incubated at 27°C and studied by light microscope every week, during 4 weeks. BM aspirate smears were stained with Giemsa and examined by an experienced parasitologist. Samples from peripheral blood or BM aspirates were collected in tubes with anticoagulant (ethylenediaminetetraacetic acid) and used as DNA sources. DNA extraction and LnPCR were performed at the National Reference Laboratory (Instituto de Salud Carlos III, Madrid, Spain). DNA was obtained using the QIAamp mini kit (Qiagen, Hilden, Germany), and LnPCR was carried out according to the methods previously published.13
Continuous variables were expressed as median and interquartile range (IQR) and categorical variables as counts and percentages. Mann–Whitney U tests were used for independent 2-group comparisons in continuous variables, and χ2 or Fisher’s exact test was used to compare categorical variables. All statistical analyses were performed using SPSS software 20.0 (IBM, SPSS, Chicago, IL).
Twenty-four children were diagnosed of VL during the study period. Diagnosis was made as follows: positive BM LnPCR, 20 children; positive blood LnPCR, 3 children and in 1 child, direct visualization of the parasite in BM.
BM LnPCR was performed in 20 children and was positive in all of them, but BM cultures for L. infantum only were positive in 30% (6 of 20) samples. Blood LnPCR was performed in 12 children and turned out to be positive in 10 (83%). Two children with negative blood LnPCR presented a positive LnPCR in BM. In 19 children with a positive LnPCR in BM, microscopic examination of BM aspirate smears revealed parasites in 21.5% (4 of 19).
Median age at diagnosis was 1.7 years (IQR: 0.77–4.62). Half of the children (12 of 24) were of Caucasian ethnicity, 11 were of African ethnicity (all born in Spain) and 1 child was of Hispanic ethnicity. At admission, 23 (95.8%) had fever, 18 (75%) splenomegaly and 18 (75%) hepatomegaly. Ten patients (41.7%) met HLH criteria within the first 96 hours from admission. Presence of HLH criteria at admission among children with and without acquired HLH is recorded in Table 1. Low hemoglobin, high fasting triglycerides, ferritin equal or greater than 500 mg/L and presence of figures of hemophagocytosis in BM were more frequent in children with acquired HLH (Table 1).
BM aspirates were performed in 20 children, 7 of them (35%) had figures of hemophagocytosis. Children with HLH criteria had lower median hemoglobin values (P = 0.002), higher ferritin (P = 0.001) and triglyceride (P = 0.001) levels and had lower fibrinogen (P = 0.04) than those with VL who did not meet HLH criteria (Table 2). There were no significant differences between children with and without evidence of HLH in terms of age, number of days of fever before treatment and days of fever since the beginning of treatment or ethnicity (Table 2).
All children were treated with liposomal amphotericin B, with a median cumulative dose of 24 mg/kg (IQR: 14–21), divided in to daily doses of 3–4 mg/kg (5 consecutive days and other doses on days 10 and 21). Two of the children received specific treatment for HLH with etoposide, cyclosporine A and dexamethasone before VL diagnosis. One girl had a relapse of VL 6 months after the first episode, and complete immunological work-up was performed including HIV status, with normal results. There were no deaths during the study period, and only 1 child was admitted to the Pediatric Intensive Care Unit with gastrointestinal bleeding because of severe hypofibrinogenemia. Two children presented persistence of fever beyond 7 days after starting VL treatment. Persistence of fever beyond 7 days was associated with the following clinical features: longer period with fever before treatment [23.5 days (IQR: 20–23. 5) vs. 13 days (IQR: 10–16), P = 0.029], lower values of hemoglobin [6.5 g/L (IQR: 6.1–6.25) vs. 7.9 g/L (IQR: 7–8. 95), P = 0.005] and higher levels of ferritin [17,301.5 μg/L (IQR: 4603–17,301.5) vs. 849 μg/L (IQR: 219.7–3082. 5), P = 0.05].
In recent years, there has been an outbreak of cutaneous and VL in Fuenlabrada, a city located in South Madrid.14 Between July 1, 2009 and December 31, 2012, 542 cases of leishmaniasis were reported in Madrid, 446 from the outbreak area. Thirty-six percent (160 of 446) were VL.14 During the study period (January 2010 and December 2011), 103 cases of VL were reported, and 24 of them were children under 16 years.14 All reported cases of children with VL were included in the study. There was a marked increase in the incidence rates from 1.5 of 100,000 inhabitants per years before the outbreak to 22.2 of 100,000 inhabitants per year during the outbreak period.14
The origin of the outbreak seemed to be related to a high prevalence (30%) of wild hares (Lepus granatensis) infected by L. infantum. In recent years, there was a disproportionate increase in the population of this rodent, presumably because of urban changes in a public park where the outbreak took place.14,15 The role of hares as reservoirs of L. infantum had not been previously reported, but its transmission from hares to sand flies has been verified by xenodiagnosis.15
About half of acquired HLH are associated with infections.4 Not only virus, particularly EBV and other herpes viruses, are the most frequent organisms triggering HLH, but also bacteria, fungi and protozoa have been involved. The etiology of acquired HLH associated to infections is highly variable depending on geographical location. For example, the majority of EBV-associated HLH cases have been described in Asia, particularly in Japan. In previous reports, all children with HLH associated to VL were from Spain and Eastern Mediterranean areas.5
In a recent study, VL-associated HLH represented 2.1% (15 of 710) of all HLH cases included in a national reference center database.5 It is thought that acquired HLH associated with VL is an uncommon complication of VL.6,7,9,11,12,16–17 A striking finding in our series was the high percentage (41%) of children who met HLH criteria. To the best of our knowledge, such a high rate of HLH associated with VL as in our series has not been previously described. We do not have an explanation for this finding. Acquired HLH is the result of an uncontrolled immune response to different stimuli.1,2,4 A different immune response according to ethnicity has been ruled out in our study, as there were no differences in this variable between children who did and did not develop HLH. A more prolonged course of Leishmania infection in children with HLH may also be discarded, as the duration of fever before starting treatment with liposomal amphotericin B was similar between the 2 groups, as was the age.
Genetic HLH is very frequently triggered by an infection, especially EBV. In these cases, differentiation between genetic and acquired HLH may be particularly difficult. Differentiating between the 2 forms of HLH is of paramount importance, because in acquired HLH due to a treatable infection, specific treatment solves the syndrome in most cases, thus avoiding unnecessary and harmful treatments. In 2 of our patients, treatment against HLH with etoposide, cyclosporin A and dexamethasone was started before VL diagnosis was made. This treatment was stopped as soon as VL diagnosis was made, but these 2 cases illustrate the difficulty to decide when and how to treat acquired HLH until causal relation with an infection is established. This fact has also been emphasized in the study performed by Bode et al5 in which 31% (4 of 13) of children with HLH associated to VL had received HLH-directed therapy before the diagnosis of Leishmania infection. Also in the article by Gagnaire et al,6 30% (4 of 12) of the patients with VL-associated HLH had an initial wrong diagnosis. These examples and our study clearly show that VL should be ruled out in people with HLH who live in or coming from endemic areas of leishmaniasis.
Clinical manifestations and laboratory abnormalities overlap not only between genetic and acquired HLH, but also in patients with VL without HLH. In both entities, there are prolonged fever, cytopenias and splenomegaly. In addition, in both conditions, there is immunoactivation and a similar pattern of cytokines, which is responsible for some of the clinical and laboratory characteristics of VL and HLH.2,18
In our series, the significant differences between children with and without HLH are listed in Table 2. Hemoglobin, fibrinogen and triglycerides were lower, and ferritin was higher in children with HLH.
Another fact that can make it difficult to differentiate between VL and genetic HLH is the age of presentation, as both conditions are more frequent in infants and young children. The lack of sensitivity (except for LnPCR) of the several diagnostic procedures for VL5 also contributes to pitfalls in differentiation between these 2 entities. According to our study and other previous studies, LnPCR in BM aspirates is the most sensitive test to diagnose VL, showing a high sensitivity (100%) in our setting.13,19 LnPCR in blood is easier to perform than BM aspiration and can be the first diagnostic step, but its negativity does not exclude the infection.
All our patients, whether or not they had HLH, had a favorable outcome with liposomal amphotericin B. Moreover, we did not find any differences in the course of the disease between patients who met HLH criteria and those who did not. This suggests that in some children with VL, secondary HLH is part of the natural history of the disease, which does not involve a poor prognosis if the infection is treated properly.
The authors thank all members of Madrid Leishmaniasis Study Group: María Isabel González Tomé, PhD, Pablo Rojo, PhD, Luis Ignacio González-Granado, MD, Manuel Paz, MD, Lucía Llorente, MD, David Andina, MD, Cecilia Pérez, MD, Ana Álvarez, MD, Saioa Jiménez, MD and Katie Badillo, MD. The authors thank Mr. Martin J. Smyth, BA, for his help in correcting the English.
1. Janka GE, Lehmberg K.. Hemophagocytic lymphohistiocytosis
: pathogenesis and treatment. Hematology Am Soc Hematol Educ Program. 2013;2013:605–611
2. Janka GE.. Familial and acquired hemophagocytic lymphohistiocytosis
. Annu Rev Med. 2012;63:233–246
3. Henter JI, Horne A, Aricó M, et al. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis
. Pediatr Blood Cancer. 2007;48:124–131
4. George MR.. Hemophagocytic lymphohistiocytosis
: review of etiologies and management. J Blood Med. 2014;5:69–86
5. Bode SF, Bogdan C, Beutel K, et al. Hemophagocytic lymphohistiocytosis
in imported pediatric visceral leishmaniasis
in a nonendemic area. J Pediatr. 2014;165:147–153.e1
6. Gagnaire MH, Galambrun C, Stéphan JL.. Hemophagocytic syndrome: a misleading complication of visceral leishmaniasis
—a series of 12 cases. Pediatrics. 2000;106:E58
7. Rajagopala S, Dutta U, Chandra KS, et al. Visceral leishmaniasis
associated hemophagocytic lymphohistiocytosis
–case report and systematic review. J Infect. 2008;56:381–388
8. Tapisiz A, Belet N, Ciftçi E, et al. Hemophagocytic lymphohistiocytosis
associated with visceral leishmaniasis
. J Trop Pediatr. 2007;53:359–361
9. Levy L, Nasereddin A, Rav-Acha M, et al. Prolonged fever, hepatosplenomegaly, and pancytopenia in a 46-year-old woman. PLoS Med. 2009;6:e1000053
10. Koubâa M, Mâaloul I, Marrakchi Ch, et al. Hemophagocytic syndrome associated with visceral leishmaniasis
in an immunocompetent adult—case report and review of the literature. Ann Hematol. 2012;91:1143–1145
11. Celik U, Alabaz D, Alhan E, et al. Diagnostic dilemma in an adolescent boy: hemophagocytic syndrome in association with kala azar. Am J Med Sci. 2007;334:139–141
12. Kontopoulou T, Tsaousis G, Vaidakis E, et al. Hemophagocytic syndrome in association with visceral leishmaniasis
. Am J Med. 2002;113:439–440
13. Cruz I, Chicharro C, Nieto J, et al. Comparison of new diagnostic tools for management of pediatric Mediterranean visceral leishmaniasis
. J Clin Microbiol. 2006;44:2343–2347
14. Arce A, Estirado A, Ordobas M, et al. Re-emergence of leishmaniasis in Spain: community outbreak in Madrid, Spain, 2009 to 2012. Euro Surveill. 2013;18:20546
15. Molina R, Jiménez MI, Cruz I, et al. The hare (Lepus granatensis
) as potential sylvatic reservoir of Leishmania infantum in Spain. Vet Parasitol. 2012;190:268–271
16. Agarwal S, Narayan S, Sharma S, et al. Hemophagocytic syndrome associated with visceral leishmaniasis
. Indian J Pediatr. 2006;73:445–446
17. Kilani B, Ammari L, Kanoun F, et al. Hemophagocytic syndrome associated with visceral leishmaniasis
. Int J Infect Dis. 2006;10:85–86
18. Goto H, Prianti Md.. Immunoactivation and immunopathogeny during active visceral leishmaniasis
. Rev Inst Med Trop Sao Paulo. 2009;51:241–246
19. Antinori S, Calattini S, Longhi E, et al. Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis
in HIV-infected and HIV-uninfected patients: a single-center, 8-year experience in Italy and review of the literature. Clin Infect Dis. 2007;44:1602–1610