• View in gallery

    District map of Nepal. Districts endemic for leishmaniasis are indicated in gray. The VL patient reported resides in Bardiya district (black), where Bheri Zonal Hospital is located. STIDH, Sukraraj Tropical and Infectious Disease Hospital.

  • View in gallery

    Agarose gel electrophoresis of the PCR-amplified DNA fragments. BD, reported patient bone marrow sample; T4 and D10, Nepalese samples infected with L. donovani; D2, negative sample for Leishmania. Marker indicates 100-bp DNA ladder.

  • 1

    World Health Organization, 2002. The World Health Report 2002. Reducing Risks, Promoting Healthy Life. Geneva, Switzerland: World Health Organization.

  • 2

    Sundar S, Mondal D, Rijal S, Bhattacharya S, Ghalib H, Kroeger A, Boelaert M, Desjeux P, Richter-Airijoki H, Harms G, 2008. Implementation research to support the initiative on the elimination of kala azar from Bangladesh, India and Nepal: the challenges for diagnosis and treatment. Trop Med Int Health 13 :2–5.

    • Search Google Scholar
    • Export Citation
  • 3

    Desjeux P, 2004. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27 :305–318.

  • 4

    Minister of Health Nepal, 2007. The Internal Assesment of Malaria, Kalaazar Control Activites 2004, 2005, 2006. Kathmandu, Nepal: Ministry of Health, Directorate of Health Service, Epidemiology and Disease Control Division.

  • 5

    World Health Organization, 1990. Control of the leishmaniases. Report of a WHO Expert Committee. World Health Organ Tech Rep Ser 793 :1–158.

    • Search Google Scholar
    • Export Citation
  • 6

    Olliaro PL, Guerin PJ, Gerstl S, Haaskjold AA, Rottingen JA, Sundar S, 2005. Treatment options for visceral leishmaniasis: a systematic review of clinical studies done in India, 1980–2004. Lancet Infect Dis 5 :763–774.

    • Search Google Scholar
    • Export Citation
  • 7

    Sundar S, Murray HW, 2005. Availability of miltefosine for the treatment of kala-azar in India. Bull World Health Organ 83 :394–395.

  • 8

    Pandey K, Pant S, Kanbara H, Shuaibu MN, Mallik AK, Pandey BD, Kaneko O, Yanagi T, 2008. Molecular detection of Leishmania parasites from whole bodies of sandflies collected in Nepal. Parasitol Res 103 :293–297.

    • Search Google Scholar
    • Export Citation
  • 9

    Karki P, Koirala S, Parija SC, Handsak SG, Das ML, 1998. A thirty day course of sodium stibogluconate for treatment of kala-azar in Nepal. Southeast Asian J Trop Med Public Health 29 :154–158.

    • Search Google Scholar
    • Export Citation
  • 10

    Rijal S, Chappuis F, Singh R, Bovier PA, Acharya P, Karki BM, Das ML, Desjeux P, Loutan L, Koirala S, 2003. Treatment of visceral leishmaniasis in south-eastern Nepal: decreasing efficacy of sodium stibogluconate and need for a policy to limit further decline. Trans R Soc Trop Med Hyg 97 :350–354.

    • Search Google Scholar
    • Export Citation
  • 11

    Sundar S, 2001. Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health 6 :849–854.

  • 12

    Seifert K, Matu S, Javier Perez-Victoria F, Castanys S, Gamarro F, Croft SL, 2003. Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents 22 :380–387.

    • Search Google Scholar
    • Export Citation
  • 13

    Troya J, Casquero A, Refoyo E, Fernández-Guerrero ML, Górgolas M, 2008. Long term failure of miltefosine in the treatment of refractory visceral leishmaniasis in AIDS patients. Scand J Infect Dis 40 :78–80.

    • Search Google Scholar
    • Export Citation
  • 14

    Sindermann H, Engel KR, Fischer C, Bommer W, 2004. Oral miltefosine for leishmaniasis in immunocompromised patients: compassionate use in 39 patients with HIV infection. Clin Infect Dis 39 :1520–1523.

    • Search Google Scholar
    • Export Citation
  • 15

    Perez-Victoria FJ, Gamarro F, Ouellette M, Castanys S, 2003. Functional cloning of the miltefosine transporter. A novel P-type phospholipid translocase from Leishmania involved in drug resistance. J Biol Chem 278 :49965–49971.

    • Search Google Scholar
    • Export Citation
  • 16

    Calvopina M, Gomez EA, Sindermann H, Cooper PJ, Hashiguchi Y, 2006. Relapse of new world diffuse cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana after miltefosine treatment. Am J Trop Med Hyg 75 :1074–1077.

    • Search Google Scholar
    • Export Citation
  • 17

    Zerpa O, Ulrich M, Blanco B, Polegre M, Avila A, Matos N, Mendoza I, Pratlong F, Ravel C, Convit J, 2007. Diffuse cutaneous leishmaniasis responds to miltefosine but then relapses. Br J Dermatol 156 :1328–1335.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Relapse of Visceral Leishmaniasis after Miltefosine Treatment in a Nepalese Patient

View More View Less
  • 1 Sukraraj Tropical and Infectious Diseases Hospital, Kathmandu, Nepal; Department of Immunogenetics, Department of Protozoology, and Animal Research Center for Tropical Infections, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan

We report the first case of visceral leishmaniasis (VL) relapse in a healthy individual after complete miltefosine treatment. The patient attended hospital with a history of fever for 2 months, splenomegaly, hepatomegaly, and weight loss. The case was confirmed as VL by microscopical detection of Leishmania parasites in a bone marrow specimen and by a positive result for the immunochromatography-based test targeting the Leishmania donovani rK39 antibody. A polymerase chain reaction (PCR) specific for the Leishmania kinetoplast minicircle gene was positive, and subsequent sequencing of the PCR-amplified product confirmed that this case was a L. donovani infection. The patient was treated with miltefosine for 28 days, during which time the response was good, and the Leishman-Donovan body (LD body) was negative on discharge. Ten months later, however, this patient again developed high fever and splenomegaly, and LD bodies and rK39 antibody were positive, thus indicating a relapse of VL. The patient was subsequently treated with 1 mg/kg of amphotericin B for a total of 14 days and recovered completely.

Visceral leishmaniasis (VL), also known as Kala azar, is a parasitic disease caused by Leishmania donovani and is responsible for about 59,000 deaths per year and 2.4 million disability-adjusted life years lost.1 India, Nepal, and Bangladesh account for 300,000 cases annually and thus suffer 60% of the global burden of the disease.2 The disease displays a wide epidemiologic diversity, which can mainly be attributed to co-infection with HIV, malnutrition, and host genetic factors.3 There are an estimated 6.5 million people at risk from the disease in Nepal, where it caused a total of 28,424 cases with 582 deaths between 1980 (when cases were first recorded) and 2006. The highest case fatality rate was recorded in 1982 (13.16%), and the highest case incidence was in 2003.4

The first line treatment of VL is a pentavalent antimonial (sodium stibogluconate [SSG]) as recommended by the World Health Organization (WHO).5 On the Indian subcontinent, the efficacy of SSG has gradually declined despite the regular increasing of both dose and duration of treatment. Current chemotherapy for leishmaniasis has been bolstered by the introduction of new drugs and formulations such as miltefosine (MLF) and liposomal amphotericin B. Although MLF is an effective oral drug, its teratogenic potential (observed in rats at a no-effect dose level of 0.6 mg/kg) and long half-life in vivo remain drawbacks.6 One of the major dangers is that its long half-life (150–200 hours) might encourage the emergence of resistant parasites in the field.7

We describe here a patient with VL infection from midwestern Nepal whose disease completely resolved clinically and parasitologically after 28 days of oral MLF (2.5 mg/kg/day) administration but relapsed 10 months later. To the best of our knowledge and available literature, such a relapse of VL in a healthy individual after complete MLF treatment has not previously been reported in Nepal.

A 19-year-old male patient from the Bardiya district (Figure 1) in midwestern Nepal was transferred to Sukraraj Tropical and Infectious Disease Hospital, Kathmandu, Nepal, from Bheri Zonal Hospital, Nepalgung, Banke, in July 2007. The patient had a high-grade fever, associated with rigor, and had experienced a loss of appetite and weight for 2 months. The patient had a history of travel to the Indian State of Uttar Paradesh, which borders the Bardiya district of Nepal. During the first month of fever, the patient was locally treated with antipyretics and taken to Bheri Zonal Hospital, the biggest government hospital in the Midwestern region, 40 km from Bardiya district. He was treated with antibiotics and anti-malarial drugs at Bheri Zonal Hospital, but after 2 weeks of continuous fever, he was transferred to Sukraraj Tropical and Infectious Disease Hospital for definite diagnosis and treatment. The patient was found to be anemic, and the spleen size was 4 cm at the first time of admission. Initial basic hematologic examination showed a white blood cell count of 6700/mm3, hemoglobin level of 8.7 g/dL, and total neutrophil count of 59%, with a lymphocyte fraction of 40%. The liver enzyme levels were moderately low but within normal range. Total serum protein level was 7%, and globulin level was increased to 5%. Random blood glucose level was 128 mg/dL, and the electrocardiogram was normal. Renal function was within normal range with blood urea of 28 mg/dL and creatinine of 0.6 mg/dL. The provisional diagnosis of leishmaniasis using rK39 (Insure; Inbios, Seattle, WA) was positive, and bone marrow aspiration was also positive for Leishman-Donovan body (LD body).

Polymerase chain reaction (PCR) analysis was performed for species identification. DNA was extracted from bone marrow smears using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) and was subjected to a PCR specific for a Leishmania minicircle kinetoplast gene. The detailed PCR process was given previously.8 Positive PCR product with the size similar to those of confirmed L. donovani samples (T4 and D10 Nepalese isolates) suggested that the case was Leishmania infection (Figure 2). The DNA fragment was recovered from agarose gel and purified using QIAquick Gel Extraction kit (Qiagen). The purified DNA fragment was directly sequenced using an ABI Prism BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) with the LIN 19 primer according to the manufacturer’s instructions.8 The obtained sequence (486 bp) showed strong homology with the L. donovani Dd8 Indian strain (MHOM/IN/DD8; accession no. Y11401), T4 isolate, and D10 isolate (98.7%, 100%, and 99.2%, respectively), supporting L. donovani infection diagnosis at the molecular level. Sequence data have been deposited in the GenBank/EMBL/DDBJ databases under accession numbers AB458388, AB458399, and AB458390.

The patient was treated with 2.5 mg/kg of MLF orally for a period of 28 days in the hospital as a directly observed therapy (DOT) and evaluated every week for the period of 28 days during the course of treatment to ensure compliance, detect any MLF-related adverse reactions, and evaluate the drug response. The patient was cured completely after 28 days of treatment as indicated by the absence of fever, impalpable spleen, and negative result for LD body.

The patient was completely free from disease for 9 months after treatment, until the development of a fever at the end of May 2008. During July 2008, the patient visited Sukraraj Tropical and Infectious Disease Hospital to investigate the cause of fever and for treatment. Physical examination showed that the patient had fever, mild anemia, and splenomegaly measuring ~4 cm in size. Routine examination including white blood cell count, erythrocyte sedimentation rate, stool, and urine was normal beside decreased hemoglobin level (7.1 g/dL). Positive rK39 test and positive LD body in the bone marrow aspiration confirmed Leishmania infection. Thus, this case was diagnosed as a relapse of leishmaniasis. After treatment with amphotericin B (1 mg/kg body weight) for a total of 14 days, the patient’s fever subsided and the spleen became impalpable. The patient has been free from disease until October 2008 when this manuscript was prepared. The patient will be observed for relapse for 1 year by the Sukraraj Tropical and Infectious Disease Hospital.

In Nepal, some data on the efficacy of SSG have been documented. Karki and others9 reported that the definite cure rate after treatment with 20 mg/kg/day SSG was 78% for 20-day treatment and 93% for 30-day treatment. In another study in southeastern Nepal from July 1999 to January 2001, treatment failure with SSG was observed in 10% of patients. 10 Treatment failure with SSG may have multiple origins—related to the drug regimen, the host immune status, and/or the parasite’s inherent tolerance itself. 11 One of the important contributing factors to the drug resistance in Bihar has been attributed to the use of infra-therapeutic doses and/or insufficient duration of SSG therapy. 11 This phenomenon also exists in Nepal. Moreover, the socio-cultural similarity and the open border between southern Nepal and northern Bihar facilitate cross-border population movements, which may also play an important role in the spread of SSG-resistant strains of L. donovani.

It has been previously shown that in vitro generation of L. donovani promastigotes resistant to MLF is possible, 12 and relapse cases of MLF-treated parasites have been observed in HIV-positive patients. 13,14 Relapse cases in HIV-positive patients suggest that host immunity plays a role in the elimination of parasites after MLF treatment. The first hints of the emergence of MLF resistance were associated with an increase in treatment failure caused by relapses, which subsequently became more common. Therefore, this report of a VL relapse after complete MLF treatment in a healthy individual may suggest an increase in MLF resistance of parasites in Nepal.

MLF is an alkylphospholipid, an analog of phosphocholine, and its chemical similarity to the natural phospholipids of cellular membranes suggests that it probably inhibits transmembrane signals and the synthesis of the cellular membrane itself. The basic mechanism for MLF resistance in Leishmania parasites consists of inactivation of the MLF transporter, LdMT. 15 Certainly, in India, the same factors as those that favor the selection of SSG resistance could also select for MLF resistance, because the drug is too expensive for impoverished patients to buy a full course of treatment and, additionally, has a comparatively long half-life. A simple mechanism for the emergence of MLF resistance is the selection of inactivating point mutations in any of the genes essential for MLF uptake. All these intrinsic features would suggest that the emergence of MLF resistance is highly likely in such areas. It should be noted that relapse cases after MLF treatment have already been reported for cutaneous leishmaniasis caused by other Leishmania species.16,17

The visceral form of leishmaniasis is endemic in the Terai region of Nepal, particularly in 13 districts of the southeast. Although this is the first reported VL case from the midwestern part of Nepal, it is difficult to confirm directly as an indigenous case because the patient was from the border district of Bardiya and has previously visited India. The patient’s family was evaluated for leishmaniasis with the rK39 test and were all shown to be negative. Thus, it remains unclear whether VL is endemic in the midwestern part of Nepal.

In conclusion, although MLF treatment induced an early clinical response in the patient, the risk of relapse indicates a need for studying new drug combinations or maintenance regimens. Because cell-mediated immunity against Leishmania parasites is poor or absent in patients with VL, combination treatments of MLF with agents that increase host immune responses, such as immunotherapy, should be considered. Policies concerning the proper use of this drug should be followed and supervised by health authorities in endemic areas of Nepal to minimize the risk of the appearance of drug failures and to ensure a long life span for this drug.

Figure 1.
Figure 1.

District map of Nepal. Districts endemic for leishmaniasis are indicated in gray. The VL patient reported resides in Bardiya district (black), where Bheri Zonal Hospital is located. STIDH, Sukraraj Tropical and Infectious Disease Hospital.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.580

Figure 2.
Figure 2.

Agarose gel electrophoresis of the PCR-amplified DNA fragments. BD, reported patient bone marrow sample; T4 and D10, Nepalese samples infected with L. donovani; D2, negative sample for Leishmania. Marker indicates 100-bp DNA ladder.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.580

*

Address correspondence to Basu Dev Pandey, Sukraraj Tropical and Infectious Diseases Hospital, Kathmandu, Nepal. E-mail: basupandey@wlink.com.np

Authors’ addresses: Basu Dev Pandey, Sukraraj Tropical and Infectious Diseases Hospital, Kathmandu, Nepal, Tel: (+977) 1 5590524, Fax: (+977) 1 4253396, E-mail: basupandey@wlink.com.np. Kishor Pandey, Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Tokyo, Japan, Tel: (+81) 358415398, Fax: (+81) 3 5841 8184, E-mail: pandey_kishor@hotmail.com. Osamu Kaneko, Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan, Tel: +81-95-819-7838, Fax: +81-95-819-7805, E-mail: okaneko@nagasaki-u.ac.jp. Tetsuo Yanagi, Animal Research Center for Tropical Infectious, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan, Tel: +81-95-819-7856, Fax: +81-95-819-7805, E-mail: f1020@cc.nagasaki-u.ac.jp. Kenji Hirayama, Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan, Tel: +81-95-819-7818, Fax: +81-95-819-7805, E-mail: hiraken@nagasaki-u.ac.jp.

Acknowledgments: The authors thank all the staff of Sukraraj Tropical and Infectious Diseases Hospital for support in the treatment. We also thank Dr. Richard Culleton for critically reading this manuscript.

Financial support: KP was supported by a scholarship from the Japanese Government Ministry of Education, Science, Sports and Culture (MONBUSHO). This study was supported by the National Bio-Resource Project (NBRP) of MEXT, Japan.

REFERENCES

  • 1

    World Health Organization, 2002. The World Health Report 2002. Reducing Risks, Promoting Healthy Life. Geneva, Switzerland: World Health Organization.

  • 2

    Sundar S, Mondal D, Rijal S, Bhattacharya S, Ghalib H, Kroeger A, Boelaert M, Desjeux P, Richter-Airijoki H, Harms G, 2008. Implementation research to support the initiative on the elimination of kala azar from Bangladesh, India and Nepal: the challenges for diagnosis and treatment. Trop Med Int Health 13 :2–5.

    • Search Google Scholar
    • Export Citation
  • 3

    Desjeux P, 2004. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27 :305–318.

  • 4

    Minister of Health Nepal, 2007. The Internal Assesment of Malaria, Kalaazar Control Activites 2004, 2005, 2006. Kathmandu, Nepal: Ministry of Health, Directorate of Health Service, Epidemiology and Disease Control Division.

  • 5

    World Health Organization, 1990. Control of the leishmaniases. Report of a WHO Expert Committee. World Health Organ Tech Rep Ser 793 :1–158.

    • Search Google Scholar
    • Export Citation
  • 6

    Olliaro PL, Guerin PJ, Gerstl S, Haaskjold AA, Rottingen JA, Sundar S, 2005. Treatment options for visceral leishmaniasis: a systematic review of clinical studies done in India, 1980–2004. Lancet Infect Dis 5 :763–774.

    • Search Google Scholar
    • Export Citation
  • 7

    Sundar S, Murray HW, 2005. Availability of miltefosine for the treatment of kala-azar in India. Bull World Health Organ 83 :394–395.

  • 8

    Pandey K, Pant S, Kanbara H, Shuaibu MN, Mallik AK, Pandey BD, Kaneko O, Yanagi T, 2008. Molecular detection of Leishmania parasites from whole bodies of sandflies collected in Nepal. Parasitol Res 103 :293–297.

    • Search Google Scholar
    • Export Citation
  • 9

    Karki P, Koirala S, Parija SC, Handsak SG, Das ML, 1998. A thirty day course of sodium stibogluconate for treatment of kala-azar in Nepal. Southeast Asian J Trop Med Public Health 29 :154–158.

    • Search Google Scholar
    • Export Citation
  • 10

    Rijal S, Chappuis F, Singh R, Bovier PA, Acharya P, Karki BM, Das ML, Desjeux P, Loutan L, Koirala S, 2003. Treatment of visceral leishmaniasis in south-eastern Nepal: decreasing efficacy of sodium stibogluconate and need for a policy to limit further decline. Trans R Soc Trop Med Hyg 97 :350–354.

    • Search Google Scholar
    • Export Citation
  • 11

    Sundar S, 2001. Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health 6 :849–854.

  • 12

    Seifert K, Matu S, Javier Perez-Victoria F, Castanys S, Gamarro F, Croft SL, 2003. Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents 22 :380–387.

    • Search Google Scholar
    • Export Citation
  • 13

    Troya J, Casquero A, Refoyo E, Fernández-Guerrero ML, Górgolas M, 2008. Long term failure of miltefosine in the treatment of refractory visceral leishmaniasis in AIDS patients. Scand J Infect Dis 40 :78–80.

    • Search Google Scholar
    • Export Citation
  • 14

    Sindermann H, Engel KR, Fischer C, Bommer W, 2004. Oral miltefosine for leishmaniasis in immunocompromised patients: compassionate use in 39 patients with HIV infection. Clin Infect Dis 39 :1520–1523.

    • Search Google Scholar
    • Export Citation
  • 15

    Perez-Victoria FJ, Gamarro F, Ouellette M, Castanys S, 2003. Functional cloning of the miltefosine transporter. A novel P-type phospholipid translocase from Leishmania involved in drug resistance. J Biol Chem 278 :49965–49971.

    • Search Google Scholar
    • Export Citation
  • 16

    Calvopina M, Gomez EA, Sindermann H, Cooper PJ, Hashiguchi Y, 2006. Relapse of new world diffuse cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana after miltefosine treatment. Am J Trop Med Hyg 75 :1074–1077.

    • Search Google Scholar
    • Export Citation
  • 17

    Zerpa O, Ulrich M, Blanco B, Polegre M, Avila A, Matos N, Mendoza I, Pratlong F, Ravel C, Convit J, 2007. Diffuse cutaneous leishmaniasis responds to miltefosine but then relapses. Br J Dermatol 156 :1328–1335.

    • Search Google Scholar
    • Export Citation
Save