Abstract
We report a case of Mediterranean visceral leishmaniasis (MVL) associated with acute lymphoblastic leukemia (ALL) from the South of Iran. The patient, a 12-year-old girl, was known to be a case of ALL and had bone pain and prolonged fever on referral. A trephine biopsy revealed a hypocellular marrow with many amastigotes. Moreover, by specific polymerase chain reaction (PCR) on peripheral blood, a 145 bp band corresponding to kDNA from the genus Leishmania was detected and the species was identified as Leishmania infantum using nested PCR. The patient was treated successfully with two courses of amphotericin B plus IFN-γ. To our knowledge this is first report of MVL/ALL from Iran and possibly the world.
Introduction
Visceral leishmaniasis (VL) is a systemic disease which is caused by parasites of the Leishmania donovani complex. The annual incidence of human visceral leishmaniasis cases worldwide is approximately 500,000 with 75,000 fatalities (Wijeyaranate et al. 1994; WHO 2000). Moreover, Leishmania–HIV co-infections in the adult population are being reported with increasing frequency (WHO 2000). The clinical signs of VL in humans include prolonged fever, hepatosplenomegaly, substantial weight loss, progressive anemia, and death (Caldas et al. 2006). Leishmania infantum is responsible for Mediterranean visceral leishmaniasis in children in the Mediterranean basin countries including Iran. The diagnosis of VL is complex because commonly occurring diseases such as malaria, typhoid, and tuberculosis have clinical features similar to VL. Moreover, some VL cases have been misdiagnosed as autoimmune hepatitis, acute lymphoblastic leukemia (ALL), and malignant lymphoma (Dalgic et al. 2005; Jones et al. 2003; Kawakami et al. 1996). Most of these misdiagnosed patients were referred from non-endemic regions where the occurrence of the disease is not expected by the physician. Additionally, atypical cells and unusual blasts may be observed in bone marrow aspirates of VL patients (Fakhar et al. 2004; Asgari et al. 2007).
Case report
In May 2005, a 12-year-old girl from Fars Province in southern Iran and known to be suffering from acute lymphoblastic leukemia was referred to Nemazee hospital in Shiraz, suffering from bone pain and prolonged fever. Peripheral blood examination showed hemoglobin concentration 7.9 g/dl; MCV, 75; MCHC, 31.6; and red blood cell morphology: hypochromia, anisopoikilocytosis, targeting and rouleaux. White blood cell differentiation: PMN 50%, Band 8%, Lymph 34%, Mono 4%, and Eos 4%. Bone marrow aspiration was done and bone marrow differentiation count included erythro 8%, pro 10%, myelo 8%, meta 20%, band 8%, seg 18%, and lymph 28%. However, trephine biopsy showed a hypocellular marrow with abundant amastigotes and containing less than 5% blast.
Five months later, the patient was again referred to the Nemazee hospital with progressive abdominal distention and fever. A physical examination revealed mild splenomegaly. Blood examination showed hemoglobin concentration of 9.1 g/dl; MCV, 70; MCHC, 22.4; a mildly decreased white blood cell count; decreased in platelet count; and anisopoikilocytosis in RBC morphology. Bone marrow differentiation count included erythro 20%, pro 2%, myelo 16%, meta 9%, band 9%, seg 15%, and lymph 29%. The myeloid and erythroid ratio was reversed. Amastigotes and megakaryocytes were observed in bone marrow aspirates and trephine biopsy but no blast was seen.
In January 2006, hematology tests showed Hb 5.5 mg/dl, mildly decreased white blood cell, decreased platelet, and WBC diff: seg 43%, band 4%, eos 1%, lymph 34%, mono 18%, and NRBC 3%. Moreover, hypochromia and anisocytosis were seen. The bone marrow was hypocellular with numerous amastigotes. However the myeloid and erythroid ratio was reversed, and megakaryocytes and amastigotes were present.
In April 2006, hematology tests showed: Hb 5.5 mg/dl, Pancytopenia (WBC 2,000 and Plt 70,000), RBC was normochromic and normocytic, differentiation of WBC included: seg 40%, lymph 54%, and mono 6%. Differentiation of bone marrow cells included: erythro 29%, pro 2%, myelo 11%, meta 12%, band 8%, seg 6%, lymph 20%, and blast cells 10%. The myeloid and erythroid ratio was reversed and megakaryocytes and amastigotes were present.
Moreover, by the specific polymerase chain reaction (PCR) on peripheral blood, a 145 bp band of kinetoplast DNA (kDNA) belonging to the genus Leishmania was detected (Lachaud et al. 2002). At the same time, using the indirect fluorescent antibody test and direct agglutination test, the titers of anti-leishmanial antibody were 1:1,024 (the titers ≥ 1:128 are considered positive) and 1:12,800 (the titers ≥ 1:3,200 are considered positive) respectively. The infecting species of parasite was identified as L. infantum by nested PCR on DNA extracted from bone marrow aspirates and preserved on microscope slides (Noyes et al. 1998). However, liver function tests were normal and the patient was treated with glucantime (60 mg/kg intramuscularly daily for 14 days). After 2 weeks, a positive response was not seen and the fever had not subsided so therapy with amphotericin B (1 mg/kg daily intravenously for 20 days) was initiated. The amphotericin B treatment was repeated with the addition of interferon (IFN-γ) (50 mg/m2 daily subcutaneously) and the patient was discharged.
Discussion
The patient was from a nomadic tribe but had settled in Fars Province where VL is endemic. The most highly endemic areas of VL in Iran are in parts of Fars and Bushehr Provinces in Southern Iran, the districts of Meshkin-shahr and Moghan in Northwest Iran and Qom Province in central of Iran, but other parts of Iran are considered as sporadic areas of VL (Asgari et al. 2006; Mohebali et al. 2005; Fakhar et al. 2004). To our knowledge this is the first report of VL/ALL from Iran and we have not found reports of this condition from any other country.
The observation of amastigotes (Leishman-donovan bodies) in bone marrow aspiration is highly specific for the diagnosis of VL but long searches may be required to demonstrate the parasite. Moreover, the absence of detectable amastigotes is insufficient to exclude leishmaniasis and serological and molecular methods should also be used (Sundar and Rai 2002).
The PCR technique has several advantages including the ability to work with small amounts of target material, the follow-up of treatment as well as the assessment of the successful cure of visceral leishmaniasis and fast detection of Leishmania in symptomatic patients and asymptomatic carriers as well as Leishmania /HIV co-infected patients (Bossolasco et al. 2003; Mary et al. 2004; Selvapandiyan et al. 2005). The most suitable target for the DNA-based diagnosis is kinetoplast DNA minicircles. The Leishmania PCR assays using peripheral blood as a clinical specimen showed to be a highly efficient non-invasive alternative with sensitivity varying from 80–100% (Fisa et al. 2002).
In our patient, therapy with glucantime failed and also there was poor response to amphotericin B. It appears that the malignancy lead to a poor response to therapy since effective immune responses have an important role in the control of the infection. HIV/Leishmania co-infections cause a similar severe disease. Accordingly, we suggest that physicians should consider the use of IFN-γ to treat this class of immunocompromised patients. Treatment and management of these cases requires patience and careful monitoring.
In endemic areas, patients with clinical signs similar to VL who are referred to a hematologist or pathologist should also be tested for Leishmania by bone marrow aspiration. Furthermore, reactivation of parasitemia and increased sensitivity to infection are more frequently observed in immunocompromised and immunosuppressed patients (Disdier et al. 1998; Èoloviæ et al. 2002).
In conclusion, we recommend that the detection of Leishmania kDNA in blood by PCR should be regarded as a valuable tool for early diagnosis as well as monitoring of patients during and after therapy instead of the conventional invasive procedures such as splenic and bone marrow aspiration. Alternatively, immunodiagnostic methods to detect antibody and antigen are useful depending upon the clinical syndrome, antigen, and the assay used.
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Acknowledgments
The authors would like to thank Dr Harry Noyes for critical review of the manuscript.
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Fakhar, M., Asgari, Q., Motazedian, M.H. et al. Mediterranean visceral leishmaniasis associated with acute lymphoblastic leukemia (ALL). Parasitol Res 103, 473–475 (2008). https://doi.org/10.1007/s00436-008-0999-z
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DOI: https://doi.org/10.1007/s00436-008-0999-z