1. In the name of GOD
Tomb of sheikh safi

2. Email: a.fathi@arums.ac.ir
Fathi . A MD
Associate professor of pediatric oncology, Ardabil university of medical sciences

3. North west of iran , Ardabil province

4. An endemic area for VL

5. Sabalan mountain

6. Old volcano

7. Hot springs

8. Hill side

9. Grassland

10. The heyran

11. Distribution of word VL

12. Hemophagocytic Lymphohistiocytosis associated with visceral leishmania
Case1 A one-year-old boy was admitted to the Bouali Hospital, Ardabil, Iran on July 2015, with a 3-weeks history of fever.
The fever was intermittent high grade .
Clinical examination revealed Hepatosplenomegaly .

13. Initial hematological examination showed :
moderate pancytopenia .
Liver function tests showed elevated levels (ALT:750 u/ml ,AST:820 u/ml)
Serological markers for EBV, HBV, HCV and HIV were negative.

14. Further investigations revealed:
Hyperferritinemia (9780 µg/dl),
Hypertriglyceridemia(TG:420 mg/dl).
Hypofibrinogenemia(50 mg/dl) .
The direct agglutination test(DAT) was positive.

15. ` The bone marrow aspirate showed :
Prominent Hemophagocytic macrophages with very low intra-and extra cellular amastigotes .

21. Case 2 An infant of 4 months of age girl was admitted to the hospital due to fever for 2 weeks.
Intermittent high grade fever persisted with anorexia and weight loss.
Clinical examination revealed :
Petechiae and Hepatosplenomegaly.
Initial hematological examination showed:
moderate pancytopenia and very severe coagulopathy (INR>8).

22. Liver function tests showed :
Elevated levels(ALT:672 u/ml,AST:1449 u/ml). ALP: 386 u/l Bill Total: (8 mg/dl Direct: 3.6 mg/dl).
Serological markers for EBV, HBV, HCV , HIV ,wright and widal were negative.
CSF was normal

23. Further investigations revealed :
Hyperferritinemia (16493 µg/dl very high ),
Hypertriglyceridemia(TG:428 mg/dl).
Hypofibrinogenemia(40 mg/l) .
The direct agglutination test(DAT) was positive.

24. But in both cases first reports was HLH with no leishman body
The bone marrow aspirate showed:
prominent Hemophagocytic macrophages with very low intra-and extra cellular amastigotes as the same of first case
But in both cases first reports was HLH with no leishman body
Due to high suspicion to Leishmaniosis ,we re evaluated the bone marrow smears and found a few intra-and extra cellular leishman bodies.

28. The laboratory and clinical criteria based on HLH diagnostic criteria were observed in our cases and diagnosis of HLH was established.
The first case died the next morning of admission due to CNS complications without any special treatment .
For second case we initiated treatment at the night of admission as soon as possible .

29. international HLH-2004 based on protocol :
Our patient was treated by intramuscular pentavalent drug glucantime 20 mg/kg/day/for 4 weeks +
international HLH-2004 based on protocol :
Etoposide and oral dexamethasone as well as IVIG ,Vit k and blood productions for patient's complications .

30. However in IAHS intense immunosuppression may have fatal consequences especially in countries with high prevalence of tuberculosis so we did not used cyclosporine A in our regimen.

31. Our patient improved clinically and hematologically in 2 weeks.
No adverse events were observed in 3 years follow up.

33. Hemophagocytic Lymphohistiocytosis
Hemophagocytic Lymphohistiocytosis (HLH) falls in two categories :
Primary (inherited/familial)
Secondary (Nonfamilial HLH)

34. Inherited disorders that can lead to HLH include :
Chediak-Higashi syndrome
Griscelli syndrome
X – linked lymphoproliferative disease (XLP)
Hermansky- Pudlak syndrome
Lysinuric protein intolerance

35. These disorders are clinically characterized by excessive , systemic production of inflammatory cytokines leading to :
Macrophage activation
Hemophagocytosis
Pancytopenia
Hepatosplenomegaly
lymphadenopathy

36. Fever
Seizures or CNS complications
Capillary leak with pulmonary insufficiency
Hypotension
Renal failure

37. The diagnosis of HLH can be established if one of this presented ( HLH-2004 guidelines):
A molecular diagnosis consistent with HLH
Diagnostic criteria for HLH (5/8) criteria

38. 1-Fever 2-Splenomegaly 3-Cytopenias(Hb<10 gr/dl,plt<100×109/dl,PMN<1.0×109) 4-Hypertriglyceridemia (>265mg/dl)and or hypofibrinogemia(≤ 150 mg/dl) 5-Hemophagocytosis in BM or spleen or lymph nods 6-Hyperferritinemia(≥500µg/dl) 7-Low or absent NK-cell activity 8-Soluble CD25 ≥ 2400 U/ml

39. Clinical Features:
The age of onset is less than 1 year in 70% .
Signs and symptoms of FHLH include :
Fever(91%), splenomegaly(98%), and hepatomegaly(94%) ( the most common early findings).
Cerebromeningeal symptoms(20%), lymph node enlargement(17%), jaundice, edema, skin rash(6%), hepatitis, hypoproteinemia, hyponatremia, VLDL↑, HDL↓,
and multisystem involvement.

40. Treatment:
The following treatment regimen , based on HLH of Histiocyte Society :
1. Dexamethasone , 10 mg/m2/day for 2 weeks follow by a decrease every 2 weeks .
2. Etoposide IV, 150 mg/m2 daily , twice – weekly for 2 weeks then weekly.

42. 3. Cyclosporine A, 3-5 mg/kg/day IV starting week 8 to reach a level of ng/ml and switching to oral of 6-10 mg/kg/day in to divided doses. 4. age – adjusted doses of IT MTX weekly for 3-6 weeks if abnormal cells persist in the CSF.

43. 5-BMT for patients with FHLH or those with persistent or recurrent Non FHLH.
The 3 –years overall survival in FHLH with this approach is 55% but 64% following HSCT.

45. Nonfamilial HLH include:
Infections associated hemophagocytic syndrome or IAHS .
Malignancy associated hemophagocytic syndrome or MAHS
With Rheumatologic disorders , Chemotherapy and immunosuppressive agents.

46. Infections associated hemophagocytic syndrome or IAHS :
The findings are similar to those in FHLH. however , decreased or absent NK cells are found more often in FHLH.

47. Infections Associated with HLH
Epstein-Barr virus
Escherichia coli
Cytomegalovirus
Salmonella sp.
Varicella virus
Enterococcus sp.
HHV6
Mycoplasma sp.
Parvovirus B19
Tick-born bacteria
Hepatitis A
Tuberculosis
HIV
Visceral leishmaniasis
Adenovirus
Plasmodium sp.
Influenza
Toxoplasma sp.
Coxsackievirus
Pneumocystis jiroveci
Torovirus
Candida sp.

48. Treatment:
Antibiotics, antiviral, antiprotozoal drugs in addition to corticosteroids and/or etoposide plus rituximab in EBV related IAHS has been used.
Patient with persistent IAHS may require the treatment of FHLH and HSCT.

49. Patient with resolved disease may discontinue therapy at 8 weeks so cyclosporine often is not in IAHS treatment protocol.

50. The microscopic diagnosis of Leishmani is difficult and repeated bone marrow studies may be required as first bone marrow is negative in 36% of cases due to pauci_microbial nature of disease in early courses.
Spleen needle aspiration biopsy has a sensitivity of approximately 98% but is associated with high risk of hemorrhage.
So rapid diagnostic tests such as DAT, IFTA and rk39 (with 97% sensitivity and 98.9% specificity) should be used.
Also the use of PCR can be helpful in the diagnosis of VL.

51. The cornerstone of the treatment of HLH associated with VL is treatment of primary disease .
In a systematic review, liposomal amphotericin B was found more successful than antimonial agents.
Few studies have reported beneficial role of IVIG in severe HLH.

53. In conclusion :
Leishmania infection must be considered in patients presenting with HLH, especially in patients living in endemic areas. Amastigotes should be intensively evaluate on bone marrow smears, with repeated sampling and use of modern diagnostic methods for early diagnosis of HLH for reducing morbidity and mortality.

54. Thank you for pay attention