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Current diagnosis and treatment of visceral leishmaniasis Expert Rev. Anti Infect. Ther. 8(8), 919–944 (2010)
Smriti Mondal1, Pradyot Bhattacharya1 and Nahid Ali†1 Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Kolkata, India † Author for correspondence: Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India Tel.: +91 332 473 3491/3493/0492/6793 Fax: +91 332 473 0284/5197
[email protected] 1
Human visceral leishmaniasis (VL), a potentially fatal disease, is most prevalent in the Indian subcontinent, East Africa and South America. Definite diagnosis and effective treatment are the primary needs for the control of VL. Diagnosis of VL has typically relied on microscopic examination of bone marrow/splenic aspirate, but serology and molecular methods are now better alternatives. The conventional drugs for treatment of VL have limitations including unresponsiveness, relapse, specific toxicities and parenteral administration lasting for long durations. Moreover, they are less effective in HIV–VL-coinfected patients. Registration of miltefosine and paromomycin, and preferential pricing of AmBisome has offered more choices for monotherapy and combination therapy for VL. Combination therapy will increase treatment efficacy and prevent the development of resistance. In addition, active case finding and vector control strategies will also have a positive impact in the control of VL. This article critically addresses the currently available diagnostic and treatment regimens for the control of VL. Keywords : anti-leishmanial therapy • combination therapy • liposomal amphotericin B • miltefosine • rK39 strip test • visceral leishmaniasis diagnosis
Leishmaniasis, a sandfly-borne diverse, complex disease, is caused by the protozoan parasite Leishmania. More than 20 species of Leishmania are transmitted to humans by approximately 30 species of phlebotomine sandflies. The disease includes several clinical syndromes (mostly cutaneous, mucocutaneous and visceral forms) that result from replication of the parasite in macrophages of the skin, nasopharyngeal mucosa and mononuclear phagocytes. The different clinical expressions and evolution of leishmaniasis depend on both parasite subtype and immune status of the host. Visceral leishmaniasis (VL) or kala-azar, the leading cause of mortality after malaria, affects poor, remote populations in 70 countries across Southeast Asia, East Africa, South America and the Mediterranean region, with 500,000 new cases of VL every year and 51,000 deaths. There is an increasing incidence of VL and spread of transmission to previously unaffected areas. The seven most affected countries – Bangladesh, Brazil, India, Ethiopia, Kenya, Nepal and Sudan – represent over 90% of new cases [201] . The Indian subcontinent alone accounts for 40% of the VL cases (nearly 100,000 of the estimated 500,000 annual cases) [202] with www.expert-reviews.com
10.1586/ERI.10.78
an estimation of 21 out of 10,000 sampled population suffering from VL [1] , and a population of 200 million at risk of infection. Eastern Africa has the second largest number of VL patients [2] and 90% of VL cases in Latin America occur in Brazil mostly in children [3] . There is a large gap between the reported and estimated cases as official data are obtained through passive case detection only, and do not include data from the private sector or nongovernmental organizations [4] . Moreover, poor surveillance systems and misdiagnosis lead to underestimation of the true incidence and case–fatality rate of VL. Studies from Sudan and India have estimated that 91% of all kala-azar deaths went unrecognized, and approximately 20% of VL patients die before their disease is recognized [2] . Visceral leishmaniasis is caused by the Leishmania donovani complex in East Africa and the Indian subcontinent, and Leishmania infantum (syn. L. chagasi) in Europe, North Africa and South America [5,6] . Transmission of VL is either zoonotic, from animal to vector to human, or anthroponotic, where transmission takes place from human
© 2010 Expert Reviews Ltd
ISSN 1476-0584
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to vector to human. Zoonotic VL is most prevalent in areas of L. infantum transmission whereas anthroponotic VL is found in areas of L. donovani transmission. The parasite selectively infects and multiplies as amastigotes in the macrophages of the liver, spleen and bone marrow. Most human VL infections run a subclinical and self-healing course [7] , but in some a fatal visceral disease evolves without treatment. The disease mainly occurs in poor communities due to malnutrition, weak immune defense systems and increased exposure to sandfly biting, especially in males and young children [8] . Mass migration and traveling to endemic areas, civil or military emergencies, famine and change in climatic conditions (floods and droughts) may accelerate the development and spread of the disease and eventually lead to epidemics [4] . VL is characterized by a prolonged fever, an enlarged spleen and liver, weight loss, progressive anemia and lowering of immunity. These symptoms occur progressively over a period of weeks or even months. Visceral leishmaniasis is sometimes followed by a chronic skin condition, post-kala-azar dermal leishmaniasis (PKDL), characterized by dispigmented macule, erythematous rashes, indurated plaques, papules and nodules. PKDL develops in 5-10% of Indian VL patients, generally after 2–7 years, and in greater than 50% of Sudanese VL patients during or within 6 months of successful antimonial therapy and occasionally in individuals without a history of kala-azar [9] . These patients serve as a reservoir of parasites, especially during interepidemic periods of kala-azar. Visceral leishmaniasis patients may be coinfected with other infectious diseases and presently HIV–VL coinfection is an emerging problem in 35 countries for the control of VL [10] . Current data show that 2–12% of all VL subjects from endemic countries [203] , and approximately 15–30% in Humera, Ethiopia [11] , are HIV–VL coinfected. In addition, there are many unreported cases from Africa or Asia due to lack of diagnostic facilities and poor reporting systems. Molecular methods have helped to determine that more than 90% of the coinfected cases are relapses rather than reinfection. HIV infection increases the risk of developing VL or clinical progression of HIV/AIDS defining conditions by 100 to 2320-times in endemic areas. In addition, it reduces the efficacy of therapeutic response and greatly increases the probability of relapse [10,12] . Because the two diseases target similar immune cells, together they exert a synergistic damaging effect on the cellular immune response and switch the immune response from Th0- or Th1-type to Th2-type with very low CD4 + counts and high viral loads (1000–1 million copies) [10] . Atypical presentations of leishmaniasis are reported in HIV patients, including visceralization of cutaneous leishmaniasis and cutaneous involvement in VL [13] . After the introduction of antiretroviral therapy (ART) for HIV, the number of coinfected cases has reduced sharply in European Leishmania-endemic countries [10,14] . However, access to ART in developing countries remains limited, leading to the spread of coinfection to other endemic areas. In addition, coinfected patients may represent an important reservoir of parasites in anthroponotic VL, with the potential to maintain infection rates in sandflies. Routine and definite diagnosis of VL still depends on the complicated invasive method of splenic or bone marrow aspirate [15] . Although several serological- and PCR-based tests have been 920
developed, their differential response depending on geographical areas requires further optimization for definite diagnosis of VL. Both HIV and Leishmania infections induce a dearth in host humoral and cellular responses that limit the diagnostic value of serological tests for coinfected patients [10] . PCR and detection of Leishmania antigens through a new latex agglutination test (KAtex [Kalon Biological Ltd, Guildford, UK]; used for urine samples) [16] are the only promising tools for diagnosis in Leishmania–HIV coinfections [17] . Treatment of VL is complex and difficult. There is a growing limitation in the availability of chemotherapeutic strategies, and emergence of resistant strains of Leishmania has aggravated the crisis for VL. All anti-leishmanial therapies are less effective in HIV-positive patients [10] . There is a high mortality rate in these coinfected patients due to concurrent illness, complications and drug toxicity as observed with the prevalent anti-leishmanial therapies [18,19] . In general, patients who fail to clear the parasites will relapse, independent of the drug used. Relapses should be treated appropriately, and combination therapy may represent the best approach to avoid drug resistance while balancing benefit with drug toxicity. Early diagnosis and treatment, although important for the VL patient, have limited impact on transmission in the absence of control of human and animal reservoirs and insect vectors. In the present article, we discuss the currently available diagnostic and treatment approaches for VL and the developments made for easy field diagnosis and safe, effective, affordable medicines for oral or parenteral administration involving shorter treatment cycles, and new drug combinations for the treatment of VL. Diagnosis of VL
The clinical symptoms of VL such as prolonged and irregular fever often associated with rigor and chills, hepatosplenomegaly, progressive anemia, lymphadenopathy, pyrexia and wasting [20] , and that of PKDL (dermal papular, macular and nodular lesions) lack specificity. Occasionally, patients may be coinfected with malaria, typhoid, TB, tropical splenomegaly, schistosomiasis and African trypanosomiasis, among others [21] . Therefore, diag nosis of VL and PKDL is complex. Highly sensitive (>95%) and specific confirmatory tests are required to differentiate between acute disease, asymptomatic infection and a cured condition, as VL is a fatal condition and the current drugs used to treat VL are toxic and not safe enough for treatment of asymptomatic infections. Moreover, such tests should be simple and affordable. Although PKDL is not life threatening, patients serve as reservoirs of parasites and contribute to disease transmission in anthroponotic VL areas. Specific diagnosis of PKDL is therefore also important. A presumptive provisional clinical diagnosis is made on the basis of presenting clinical features and history of living in an area endemic for VL. Routine laboratory tests reveal pancytopenia and hypergammaglobulinemia as common manifestations of VL. Hypergammaglobulinemia can be detected by Formol gel test (FGT) or aldehyde test. Owing to its low sensitivity, FGT is not widely accepted. Expert Rev. Anti Infect. Ther. 8(8), (2010)
Current diagnosis & treatment of visceral leishmaniasis
Microscopic observation of Leishmania parasites
Microscopic observation of the amastigote form of Leishmania parasite in bone marrow, spleen or lymph node aspirate of VL patients remains the most reliable and confirmatory method for diagnosis of kala-azar. Presence of amastigotes can be confirmed by staining the smears with Giemsa’s stain, giving the cytoplasm pale blue and the nucleus and kinetoplast purple–pink coloration [21] . Bone marrow aspirates are obtained from sternal or iliac crest puncture and splenic aspirate by inserting a fine needle into the subcutaneous tissue over the area of the spleen below the costal margin [22] . Lymph node aspirates are collected by puncturing inguinal lymph nodes [23] by ultrasound-guided fine needles [24] . Although bone marrow aspiration is safe and less painful than spleen or lymph node aspiration, sensitivity and specificity (both >90%) of splenic aspirate is more than that of bone marrow (53–86%) and lymph node aspirates (sensitivity 53–65%) (Table 1) [22,25] . Parasite density can also be estimated by this method. However, a few reports suggest that parasitological diagnosis can miss one-fifth of the patients if it is used as the sole diagnostic criterion [26] . Moreover, due to the risk of fatal hemorrhage (~0.1%), splenic aspiration requires considerable technical expertise and should be carried out in patients with a high platelet count [21] . Antibody detection tests
Serological tests that have been developed to replace parasito logical methods for the diagnoses of VL in the field are noninvasive and highly sensitive. VL is marked by high levels of Leishmania-specific serum antibodies that appear soon after infection [21] . The elevated antibody titers against promastigote or amastigote antigen, their fractions or recombinant antigens have been extensively exploited for specific serodiagnosis of VL. Several tests that detect specific anti-leishmanial antibodies include indirect immunofluorescence assay test (IFAT), direct agglutination test (DAT), ELISA, dot ELISA, immunoblot and strip test. Comparison of several serological tests, DAT-FD, rK39 and rK26 strip test, and antigen detection through KAtex, in Bihar, India, showed that DAT-FD and rK39 strip test were highly sensitive and performed better than parasitological diagnosis though rK26 strip test, and that KAtex did not show satisfactory results [26] . Reproducibility of the serological tests between field and central laboratories was excellent. Indirect immunofluorescence assay test
Indirect immunofluorescence assay test is one of the most commonly used tests for detection of anti-leishmanial antibodies in both blood and serum. It uses the fixed promastigotes as antigen and detects antibodies that are established in the very early stages of infection and are undetectable 6–9 months after cure. IFAT has been carried out in different endemic regions such as Brazil, Spain, Tunisia, Italy and Iran. The sensitivity of the tests varies. While some reports suggest a sensitivity between 28.5 and 86.6% [27] , higher sensitivity (>80%) and specificity (>90%) have been reported by others (Table 1) [28–30] . There is a possibility, however, of cross-reaction with trypanosomal sera in these regions [27] . Use of Leishmania amastigotes as antigen may increase the sensitivity www.expert-reviews.com
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of the test. IFAT is also poorly adapted to field conditions. Direct fluorescent antibody test is used to detect the amastigote antigen in the tissue sections or smears with the help of fluorescent dye-conjugated antibodies used as tracers. This test is more useful in the diagnosis of cutaneous leishmaniasis, mucocutaneous leishmaniasis and PKDL. Horse radish peroxidase-conjugated antibody can also be used in place of fluorescence to avoid the requirement of a fluorescence microscope. Moreover, the stained slides can be stored for a long time. Direct agglutination test
Direct agglutination test is a semiquantitative test that uses microplates with V-shaped wells in which increasing dilutions of patient’s serum or blood is mixed with stained killed promasti gotes of L. donovani. If Leishmania-specific antibodies are present, agglutination occurs within 18 h, which is visible with the naked eye. The end point titer is taken as the last well where agglutination is observed. The test has been validated in several endemic areas such as Sudan, Ethiopia, Kenya, Brazil, India, Nepal and Bangladesh, and is being used for diagnosis of VL. A meta-ana lysis including 30 studies showed sensitivity and specificity estimates of 94.8 and 97.1%, respectively (Table 1) [31] . Recent studies on DAT showed sensitivity and specificity of approximately 95% for Indian VL patients [26,32,33] . Similar results were also observed in Nepal [32] , Brazil [29,34] , Ethiopia [32,35] , Kenya [32] , Venezuela [36] and Syria [37] with high sensitivity and variable specificity. A relatively low sensitivity of less than 90% was observed in Sudanese VL patients [32] and a mixed response was observed in VL patients from Iran [38] . The overall presentation of DAT as a diagnostic tool for VL is satisfactory and economic (US$1.5–2.5 per test) [26] . The performance of DAT is independent of the geographical region or Leishmania species, however, freeze-dried antigen performed better than liquid antigen [39] . Use of DAT is limited especially at rural centers due to the requirement for laboratory settings, well-trained technicians, storage of the antigen at 2–8°C once it has been dissolved, prolonged incubation and regular quality control. To overcome the drawbacks of DAT, a fast agglutination-screening test (FAST) has been developed, which can rapidly detect (90
>90
[22]
Pro Ag of La
83.3
97.6
[146]
Li Ag
80.3
90.5
[147]
Pro Ag of Li
96
ND
[28]
Dried Lc pro
100
100
[44]
Freeze-dried Ld Ag
85.7–100
62.3–98.8
Glycerol – preserved Ag
86.7
92.1
[148]
Formaldehyde fixed Ag
89.5
92.6
[148]
Ld liquid Ag
91
96.6
[44]
Leishmania Ag
100
55
[63]
Freeze-dried Ld Ag
91–100
85–100
Ags from Li
70.5
100
Aqueous Ag
97
87
Ld Ag
90.7–98
69–100
Freeze-dried Lc Ag
96.6
53.3–100
[43]
Ld Ag
100
97.8
[41]
Pro Ag
100
92.5–94.7
– Africa
Ld Pro Ag
93
ND
– Asia
Crude soluble Ld pro Ag
80, 95.7
72, 87.7
Crude Li Ag
83.6
90.5
[43]
Li Ag
83.6
90.5
[148]
Purified Li Ab
60
91.2
[43]
LAg
93.3–100
85–97.3
rLmSODB1
72.7
97.6
rKRP42
94, 94.6
99.6, 99.3
rK9
78
84
rK26
38, 96.8
80, 100
[48,49]
rK39
100
96.2–100
[47,48]
Indirect methods Antibody-detection tests • Indirect immunofluorescence test
• Direct agglutination test – Africa
– Asia
– South America • Fast agglutination screening test
[32,35,40,69]
[26,32,37] [37] [149] [44,150]
[37,41]
• ELISA [45] [48,49]
[33,44,46] [151] [42,48] [48]
Clinical symptoms: high fever, rigor and chills, hepatosplenomegaly, hypergammaglobulinemia, progressive anemia and pancytopenia. Ab: Antibody; Ag: Antigen; Am: Amastigote; La: Leishmania amazonensis; LAg: L. donovani promastigote membrane antigen; Lc: Leishmania chagasi; Ld: Leishmania donovani; Li: Leishmania infantum; ND: Not done; Pro: Promastigote.
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Table 1. Diagnostic methods for visceral leishmaniasis (cont.). Method
Antigen/antibody used
Sensitivity (%) Specificity (%)
Ref.
Lc Pro lysate
98
69
[55]
Lc Am lysate
99
76
[55]
Pro Ag
50
97.6
Lc Pro Ag
88, 89.8
85, ND
rLdccys 1
80
96
[55]
‘Q’ protein
100
43
[44]
KMP11
100
ND
[44]
rH2A
100
100
[44]
rK26
66.7
100
[146]
rK39
69.4
100
[146]
rK39–IgG
88.6
ND
[29]
rLepp12
100
100
[53]
14, 18 kDa
ND
ND
[54]
30 kDa, rLdccys 1 from Lc
100
ND
[55]
31, 34, 51, 63, 72, 91, 120 kDa from Ld
73–100
100
[52]
– Africa
rK39
71.7–96
70–99
– Asia
rK39
82.4–100
71–100
rK26
21.3
100
rK39
85.7–100
96–100
[44,155]
– Africa
57.7–90.2
64–100
[32,69]
– Asia
35.8–100
87.6–100
– South America
81.4
100
• PCR
73.2–100
87.2–100
• Real-time PCR
100
ND
[68,159] [79,160]
Indirect methods (cont.) – South America
• Immunoblotting assay
[146] [29,152]
• Immunochromatographic strip test
– America
[32,35,59,69,153] [26,27,32,33,37,48,58,60,154] [26]
Antigen detection test • Latex agglutination test
[25,29,32,38,44,71,156] [44]
Molecular methods
• PCR–ELISA
83.9
100
• PCR–restriction fragment length polymorphism
100
ND
[28,68,73,76,79,157,158]
[68]
Clinical symptoms: high fever, rigor and chills, hepatosplenomegaly, hypergammaglobulinemia, progressive anemia and pancytopenia. Ab: Antibody; Ag: Antigen; Am: Amastigote; La: Leishmania amazonensis; LAg: L. donovani promastigote membrane antigen; Lc: Leishmania chagasi; Ld: Leishmania donovani; Li: Leishmania infantum; ND: Not done; Pro: Promastigote.
sensitivity and specificity in India, Brazil, Spain and Sudan. A less sensitive response was observed in Iran [38,43] . Similar studies with recombinant gp63, rK39, r gene B protein (rGBP), r-histone proteins H2A and H2B, rLACK, rP20, rPSA-2 and purified lipophosphoglycan showed good performance for the serodiagnosis of Mediterranean VL [47] . rK39 showed the best specificity (97%) and the best predictive value of the positive test (92%) [44,47] . Other antigens that have been used are rGBP [21] , www.expert-reviews.com
rK9, rK26 [48] , 200 kDa L. donovani amastigote antigen, rA2, rH2A, KMP11 and Q protein (Table 1) [44] with varying degrees of sensitivity and specificity. Serum and urine-based rK42 ELISA and chimeric antigen K9–K39–K26 showed very high sensitivity and specificity [42,48] . Ld-ESM antigen released by L. donovani promastigotes into the protein-free medium has also been used for the serodiagnosis of VL by ELISA [44] . The more frequently used recombinant antigens are rK39 and rK26. rK26 ELISA 923
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yielded even better results than DAT [49] . rK39 is highly sensitive and predictive for onset of acute disease [21] . However, reports from Brazil and Argentina revealed decreased sensitivity for rK39 and rK26 tests, although specificity was 100% [50] . In Sudan, the rK39 ELISA test is reported to miss 7% of parasitologically proven cases [51] . ELISA is a useful laboratory diagnosis tool for VL and to screen a large number of samples at a rapid pace, but its field application is limited due to the requirement of trained technical expertise and laboratory instruments. Immunoblotting
This method involves separation of leishmanial antigens by SDS-PAGE followed by western blotting with serum samples from the patients. Serodiagnosis using immunoblotting of soluble antigens has been reported to be highly sensitive and specific. The band patterns can correlate with disease stages [21] . The commercially available electrochemiluminiscent kit enhances its sensitivity several fold. Seven polypeptides of L. donovani promastigote antigen of approximate molecular weights of 31, 34, 51, 63, 72, 91 and 120 kDa were recognized by all kala-azar patient sera tested [52] . Patient samples were also tested with other antigenic subunits, rLepp12 [53] , 14 and 18 kDa [54] , and 70, 56, 17, 90, 14 and 30 kDa [55] . Absence of antibody for a 31-kDa band of promastigote antigen in PKDL sera in comparison to the majority of the VL sera both before and after treatment, suggested the differentiation of PKDL from past and ongoing VL infection through immunoblotting [46] . Despite high sensitivity and specificity, immunoblotting can only be performed for laboratory diagnosis. rK39-based immunochromatographic test
Since rK39 proved to be most accurate when used as an ELISA assay, it was developed into an immunochromatographic test (ICT) strip or dipstick format that was more simple and suitable for field use. rK39 ICTs are easy to perform, rapid (10–20 min), economic (~US$1 per test) and provide reproducible results. Both the sensitivity and specificity of this test are high in most of the endemic regions, especially in the Indian subcontinent (Table 1) [37,56–59] . A meta-analysis with 13 validation studies of the rK39 ICT showed sensitivity and specificity estimates of 93.9 and 95.3%, respectively [31] . This test is quite effective in diagnosing PKDL [56,60] , with 95% sensitivity in polymorphic presentation and 73% with macular PKDL cases in India [61] . The rK39 strip test was also used in the diagnosis of HIV–VL coinfection, which showed 77% sensitivity [35,57] . The same test when employed with sputum samples yielded satisfactory results [62] . Recently, the excellent diagnostic performance of rK39 ICT was confirmed in India, Bangladesh and Nepal [26,56] ; however, this test proved to be less accurate in East Africa [32,35,63] . Recent studies carried out in East Africa and Asia showed better performance with DiaMed Opti-Leish IT (DiaMed AG, Switzerland), a newly manufactured rK39 dipstick, than with other dipstick formats in East Africa [56,59,64] . Unfortunately, two different rK39 dipstick formats showed poor sensitivities of 20% (Corixa/IDRI, WA, USA) and 22% (ACON Laboratories, PA, USA), in the diagnosis of European HIV–VL-coinfected patients [10,65] . Recently, use 924
of rapid tests including IT-LEISH (DiaMed AG) for the detection of rK39 antigen [66] and SIGNAL-KA (Span Diagnostics Ltd, Surat, India) for rKE16 antigen [67] have been reported. Both these tests exhibited high sensitivities (93 and 92.3%, respectively) and specificities (97 and 99%, respectively). The rK26 strip test detected only a fifth of active patients and, thus, was not useful in the diagnosis of Indian VL [26] . Antigen detection test
As serological tests cannot readily distinguish between current, subclinical and past infection, antigen detection tests may provide a better means of diagnosing VL. Antigen levels directly correlate with parasite load, being high during disease and downregulated at cure and, therefore, can determine disease progression or cure. Antigen detection may be an ideal test in immunocompromised patients where antibody response is not satisfactory. However, detection of antigen in patient’s serum may be complicated owing to the presence of antibodies and immune complexes, for example, which may mask the immunologically important antigenic determinants or competitively inhibit the binding of free antigen [68] . Latex agglutination test (KAtex)
KAtex is a simple, easy-to-perform, inexpensive, field applicable, rapid test, which can be performed without laboratory facilities. This test is based on the detection of leishmanial antigen (lowmolecular-weight 5–20-kD glycoconjugate antigen) in the urine of VL patients [25] . Latex particles are added to a small amount of boiled urine and the degree of agglutination is observed. The test showed a sensitivity of 68–100% and specificity of 100% using the urine collected from confirmed VL patients and controls from Brazil, Yemen and Nepal [25] . Other studies reported sensitivities and specificities of 95.2 and 100% in Sudan, 67 and 99% in India, 57 and above 90%, and 47.7 and 98.7% in two studies in Nepal, 73.9 and 82.4% in Ethiopia, and 82.7 and 98.9% in Iran (Table 1) [25,26,32,69,70] . A sensitivity of 85.7% was observed in HIV-coinfected patients and may be used for primary diagnosis and monitoring treatment efficacy in these patients [16] . KAtex correlated well with cure in a high proportion (97–100%) of patients during anti-leishmanial treatment [25] . The major drawbacks of this test are low sensitivity and the need to boil the urine or treat it with dithiothreitol before the test to remove false-positive reactivity [71] . Boiling also limits field applicability of the test. Molecular methods PCR
PCR is one of the most sensitive and specific methods for clinical diagnosis of VL that amplifies the parasite DNA by targeting specific conserved regions of the Leishmania gene. PCR-based assays offer an alternative for the demonstration of parasites in clinical samples [21] . The test allows highly sensitive and specific (up to 100%) detection of the Leishmania parasite irrespective of species or genus [68,72] . Furthermore, because of the short half-life of DNA in the body (24 h) [73] , PCR indicates the current (or very recent) presence of living parasites. In addition, unlike ELISA, no host species-specific reagents are required and the same reagents can be Expert Rev. Anti Infect. Ther. 8(8), (2010)
Current diagnosis & treatment of visceral leishmaniasis
used for specimens from humans, dogs or any other animal host [68] . PCR assay of different biological samples in kala-azar patients has been investigated for assessment of cure and prediction of treatment outcome [28,73] . Bone marrow, lymph node aspirates, skin biopsies, skin scrape exudates, blood/serum samples and peripheral blood mononuclear cells have been exploited for PCR in different studies [68,72] . PCR can also be carried out by using material spotted on filter paper and from Giemsa-stained slides [68,72] . This sampling method is less invasive, thus providing the opportunity for easy follow-up examination. Various gene targets including internal transcribed spacer 1 (ITS1), minicircles of kinetoplast, miniexon regions, 18S rRNA, small subunit rRNA genes, b-tubulin gene region, gp63 gene locus [21] , specific genomic sequences (457–927 bp) of DNA [74] and cytochrome b (cyt b) genes [75] are in use. PCR has also been used to diagnose PKDL [74] and HIV–VL coinfection [10] with satisfactory results and PCR has been carried out with urine samples [76] and buccal and conjunctival swabs [21,77] , all of which exhibit high sensitivity and specificity. Other modified forms of PCR such as differential display reverse transcriptase-PCR [78] , real time-PCR [10,72] and PCR–ELISA [79] are found to be more sensitive than conventional PCR. Despite its promising features, PCR is restricted to well-equipped laboratory settings and certain hospitals, partly due to technical complexity involving DNA extraction, and PCR and product detection. A few studies have reported low sensitivity of the PCR method, which might be due to low parasite load or temporary absence of parasites from the blood of VL patients [80] . Other recently used molecular techniques include SMART Leish Real-Time PCR kit (collaboration between the US Army and Cepheid, Inc., CA, USA) to diagnose cutaneous leishmaniasis [81] , reverse dot blot PCR to diagnose old world leishmaniasis [82] and cellulose acetate electrophoresis to identify any known species of Leishmania [83] . All three techniques are highly sensitive and specific, and offer new avenues in the diagnosis of the disease. Leishmania-nested PCR has been assessed for diagnosis of kala-azar and as a test of cure among kala-azar patients in Bangladesh [80] . Restriction fragment length polymorphism of the PCR products helps to identify different Leishmania species. Recently, a Leishmania-specific PCR-oligochromatographic test (Leishmania OligoC-TesT) has been developed that targets the Leishmania 18S rRNA gene and provides a simple and rapid dipstick format for detection of PCR products [84] for field application. FISH using peptide nucleic acid probes (PNA FISH) is a highly accurate and cost-effective molecular technique that has recently been applied for diagnosing trypanosomiasis [68] and could be used for diagnosis of VL. VL treatment
In the absence of any vaccine, anti-leishmanial chemotherapy is the only option for the control of VL. However, there is only a limited array of drugs for VL owing to lack of commercial incentive and involvement of public health-oriented R&D. Until the year 2000, pentavalent antimonials (Sbv) was the mainstay of treatment for VL. However, the drug has developed resistance in parts of northern Bihar. Since 2000, other available alternatives were amphotericin B (AmB), lipid formulations of AmB, www.expert-reviews.com
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miltefosine and paromomycin, all of which have limitations in the form of toxicity, variable efficacy, price and/or inconvenient treatment schedules. All are parenteral except miltefosine, which is orally administered. Besides these drugs, there are few new chemical entities that may enter clinical trials in the coming years, and we anticipate dependence on these limited drugs for the next 5–10 years. A desired VL treatment should be highly effective, preferably short-course, easily administered in the outpatient setting with no known resistant strain, with increased efficacy in HIV coinfection, affordable and safe. The Institute of OneWorld Health (iOWH) and Drugs for Neglected Diseases Initiative (DNDi) together with the WHO have initiated renewed research and drug development for neglected diseases. Current therapeutic modalities Pentavalent antimonials (Sbv)
Worldwide, Sbv has been the first-line treatment for VL for the last 70 years. Organic Sbv, urea stibamate, was first used in 1912. It was later replaced by sodium stibogluconate (SSG; Pentostam) and a few years later by meglumine antimoniate (Glucantime) for the treatment of VL (Table 2) . However, due to the high cost (~US$200 per patient), branded SSG was unavailable to patients in some developing countries and was mainly used in Europe, Africa and South America. Later, in the 1990s, a generic sodium antimony gluconate (SAG; Albert David Ltd, India; US$50 per patient) was used to treat patients adequately without any significant difference in final cure rate [85] . SAG is now widely used in India, Bangladesh, Nepal, Sudan and Ethiopia. In 2005, the price of Glucantime was also considerably reduced for less-developed countries and nongovernmental organizations for the treatment of VL [4] . Initially, the drug was used at a very low dose (10 mg/kg per day for 6–10 days) and was well tolerated [86] . When failures started to occur in Bihar, drug dose and duration of therapy were gradually increased to combat resistance. The increased drug dose (20 mg/kg, 600 mg total) for 20 days or longer was 83–86% effective [86,87] . In 1982, the WHO recommended Sbv at 20 mg/kg (maximum 850 mg) for 20–30 days in new cases and for double duration (40–60 days) in relapse cases [87] . Since the mid-1990s, a regimen of 20 mg/kg/day for 30 days cured only 36–69% of the cases demonstrating the progressive downfall of Sbv treatment in Bihar [88] . However, the drug continued to be effective in other endemic areas. In Bangladesh, a regimen of Sbv, 20 mg/kg for 20 days is used and may still be effective, although follow-up of patients to detect final cure is not conducted regularly [89] . Throughout Sudan, cure rates of 95% or higher have been consistently obtained with a standard 30 days regimen of Sbv [90] with no trend towards the development of resistance. For the treatment of PKDL in India and Bangladesh, Sbv is administered in 20 mg/kg intramuscular or intravenous injections for 120 days. Intramuscular administration is extremely painful as the irritant drug has to be injected in large volumes. The intravenous route is less painful but impractical for field application to treat large numbers of patients. Sbv is highly toxic with fatal side effects including cardiac arrhythmia and acute pancreatitis, more evident in HIV–VL-coinfected patients [19] . Increased drug dose directly 925
926 >95% in all regions
Not reported
~60% (Bihar, India)
Cardiac arrhythmias, pancreatitis, nephrotoxicity, hepatotoxicity
First-line treatment for VL in all Sbv High efficacy; responsive regions; relatively no resistance reported cheap and less toxic than AmB; daily injections favors outpatient treatment
Resistance
Toxicity
Advantages
Oral
50–100 mg (for bodyweight of 98%; single dose 94–97% (India) (5–15 mg/kg) 91–100% cure established only in India. Treatment failure in Sudan with 20–30 mg/kg in a few cases
iv.
5–20 mg/kg total dose in 1–10 doses over 1–20 days
Brazil (Ethiopia, Kenya, Sudan and India not registered but allowed for use on compassionate basis)
Gilead (AmBisome)
Liposomal amphotericin B
Exchange rates for pricing of drugs at [207]. † Estimated weight of an average Indian/African VL patient is 35 kg. ‡ Sources and prices at [208]. AmB: Amphotericin B; im.: Intramuscular; iOWH: Institute for OneWorld Health; iv.: Intravenous; SSG: Sodium stibogluconate; VL: Visceral leishmaniasis. Data taken from [95].
Mainly nephrotoxicity, infusionrelated fever and chills
iv.
0.75–1 mg/kg for 15–20 infusions either daily or on alternate days (15–20 mg/kg total dose)
36–95% (depending on the geographical region)
20 mg/kg daily for 20–30 days (depending on the geographical region)
Regimen
Brazil, Ethiopia, Kenya, Sudan, India, Nepal and Uganda
Clinical efficacy
Bangladesh, Brazil, Ethiopia, Kenya, Sudan, India, Nepal and Uganda
Registration
Sarabhai Piramal Pharmaceuticals, India; Lifecare Innovations Pvt. Ltd., India (Fungitericin); Bristol-Myers Squibb (Fungizone); Cobinopharm, Spain; other generic companies
Amphotericin B
Administration iv. or im.
Albert David, India (generic SSG); Wellcome (Pentostam); Sanofi Aventis (meglumine antimoniate; Glucantime)
Marketing authorization
Pentavalent antimonials
Table 2. Currently available drugs for the treatment of visceral leishmaniasis.
Cheapest drug; few side effects; daily im. injections can be given as ambulatory care
Nephrotoxicity, ototoxicity and hepatotoxicity (all relatively rare)
Laboratory isolates
94% (India)
im.
15 mg/kg for 21 days (India only)
India
Gland Pharma/iOWH
Paramomycin
Review Mondal, Bhattacharya & Ali
Expert Rev. Anti Infect. Ther. 8(8), (2010)
Pentamidine
Exchange rates for pricing of drugs at [207]. † Estimated weight of an average Indian/African VL patient is 35 kg. ‡ Sources and prices at [208]. AmB: Amphotericin B; im.: Intramuscular; iOWH: Institute for OneWorld Health; iv.: Intravenous; SSG: Sodium stibogluconate; VL: Visceral leishmaniasis. Data taken from [95].
Approximate cost of drugs per course for a 35 kg† VL patient (US$)
Review
correlates with increased toxicity. As ‘bad’ batches have caused fatal cardiotoxicity in the past, caution must be exercised before using Sbv from new manufacturers. In two reports from India and Nepal, a high incidence of fatal cardiotoxicity was reported with use of antimony made from an unknown manufacturer [86] .
~$10 for adults and ~$5 for pediatric treatment Preferential price: $70–280 (for 5–20 mg/kg total dose) Commercial price: ~ten-times the aforementioned costs
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SSG (generic) ~$35–53; Pentostam ~$140–210; Glucantime ~$39–59 (preferential price; based on 20–30 day course)
Generic price: ~$26.88–35.84 (Cobinopharm‡; 15–20 mg/kg total dose) Price varies for other generic manufacturers
Preferential price: ~$61.5 and $84.5 or, if >75,000 packs ordered, $53.8 and $71.7, respectively, for 50–100 mg daily dose for 28 days Commercial price: ~$75–150
Low efficacy in Sudan; prolonged treatment; potential for resistance Relatively expensive; low compliance; possible teratogenicity; potential for resistance Expensive; requires slow iv. infusion heat stability (stored 95%) in VL patients in India [86,96] . Primary resistance and relapses are uncommon. Occasional relapse (1%) has been reported, which can be treated successfully with the same drug [86] . Development of PKDL has reduced significantly in Bihar after AmB treatment of VL patients [97,98] . Despite a high cure rate, AmB is a toxic drug that requires intravenous administration of long duration, and has frequent adverse effects, including infusion-related fever and chills, nephrotoxicity and hypokalemia [25,96] , requiring hospitalization of patients for 4–5 weeks. AmB is registered for kala-azar treatment in all VL-endemic countries other than Bangladesh [95] and is currently the drug of choice in India [86] . In Uganda, AmB demonstrated a similar effectiveness and safety profile as that of meglumine antimoniate [99] , and is presently used as a second-line drug in East Africa [100] . However, 927
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it is unsuitable for use in interior remote areas where hospital facilities are lacking, limiting the number of patients that can be treated at a given time. Lipid formulations of AmB
To overcome the drawbacks of AmB, less toxic and more effective lipid formulations of AmB have been developed with the replacement of deoxycholate with lipids. These formulations lead to targeted drug delivery to macrophages of the spleen, liver and bone marrow without compromising the efficacy of the drug. It is possible to deliver higher doses of drugs over short periods [86] . The effective dose requirement varies from region to region. These formulations include liposomal AmB (AmBisome; Gilead Sciences, CA, USA), AmB colloidal dispersion (ABCD [Amphocil]), AmB lipid complex (ABLC [Abelcet]) [101] and Fungisome (Lifecare Innovations, India) [102] . These preparations have been tested successfully in VL in India, Kenya, Brazil and Europe, where HIV-coinfected individuals were also included [102] . AmBisome is an intravenous infusion with the highest therapeutic index among all the liposomal formulations of AmB and is currently the first-line drug for VL in Europe and the USA. It has rapid and up to 98% cure rates with a total drug dose of 18–21 mg/kg delivered over a short duration [103] . Because of its minimum adverse effects and increased tolerability, AmBisome has been used in basic field conditions in Sudan [86,103] and Ethiopia [104] . Various AmBisome regimens have been tested in Bihar ranging from 3.75 to 15 mg/kg total dose, which showed 89–100% cure [103] . Dosing is normally fractioned through five doses but a single infusion of 5, 7.5 and 15 mg/kg has showed a high success rate and limited adverse effects [103] . Safety and efficacy of AmBisome at a total dose of 20 mg/kg administered over 10 days in Bihar was highly tolerable with initial cure rates over 98% [105] . In a comparative study, single-dose AmBisome 10 mg/kg showed efficacy of approximately 96% cure similar to 15 alternate-day infusions of AmB (1 mg/kg; conventional therapy) in northeastern India [106] . However, in Sudan, treatment failures have been observed with AmBisome monotherapy, even when total doses exceed 20–30 mg/kg [107] . Lack of efficacy was also observed in HIV-coinfected patients in Ethiopia with greater than 50% treatment failure in relapsing patients [104] . AmBisome administered at 2.5 mg/kg/day for 20 days, however, completely cleared the skin rashes in 83% of PKDL patients in Sudan who did not respond to repeated SSG treatment [108] . Initially, due to the prohibitive commercial price of AmBisome (US$200/50-mg vial) in poor-resourced endemic countries, even short courses of AmBisome were still more expensive than conventional AmB treatment because of the low hospitalization costs in these countries [86] . Recently, the price of AmBisome has been reduced from US$200 to US$20/50-mg vial for use in VL, first with the charity Médecins Sans Frontières (MSF) and more recently with the WHO for developing countries (agreement between Gilead and WHO of 14 March 2007) [95] . With this preferential pricing, single-dose AmBisome treatment (10 mg/kg) is cost effective compared with conventional AmB treatment [109] . The effective dose regimen of AmBisome varies from region to region. According to 928
the latest WHO recommendations, 15 or 10 mg/kg AmBisome regimens may be used in Southern Asia whereas 20 mg/kg may be used elsewhere [204] . Care should be taken not to extrapolate the results of carefully conducted clinical trials to clinical practice under field conditions, nor from one region to another [4] . Among other lipid formulations, Abelcet has been evaluated for re-treating Sbv failures in India. A total dose of 10–15 mg/kg showed a cure rate of 90–100% [86] . In HIV–VL-coinfected patients in Europe, its efficacy was limited to 33–42%. However, for prevention of relapse, Abelcet, administered at 5 mg/kg every 21 days for 12 months, was useful as secondary prophylaxis in 50% of HIV-infected patients, and was well tolerated [110,111] . Amphocil was first used in Brazil at a total dose of 10 and 14 mg/kg and showed a cure rate of 90 and 100%, respectively [112] . In a larger clinical study in India, Amphocil was evaluated in different doses of 7.5, 10 and 15 mg/kg which showed 96–97% cure at 6 months post-treatment [113] . Amphomul (Bharat Serums and Vaccines, India), a new AmB emulsion, has proved highly effective and safe in a small study of VL patients in three different short-course dosing regimens [114] . Subsequently, Phase II studies with different short-course regimens of Amphomul (total dose 15 mg/kg) were carried out (Table 3) . Single-dose Amphomul (15 mg/kg) was found to be highly effective and safe for the treatment of VL [115] . Fungisome is another liposomal AmB formulation that was found to be safe and effective in VL when administered at total doses of 14–21 mg/kg and showed cure rates of 90.9–100% [116] . At a lower dose of 10 mg/kg the same formulation showed 90% cure in Indian VL patients [117] . Fungisome is now being studied for higher dose regimens of 15 mg/kg (7.5 mg double-dose) (Table 3) . However, none of the aforementioned lipid formulations have been compared together in a clinical trial. Miltefosine
Miltefosine (hexadecylphosphocholine) is an alkylphospholipid derivative and was developed as an anticancer drug. Its anti-leishmanial efficacy is considered as a landmark event as it is the first effective oral treatment for VL, including Sbv-resistant cases [118] . These findings led to clinical trials and registration of miltefosine in India in March 2002 for oral treatment of VL. The recommended daily dose of miltefosine is 50–100 mg (for a bodyweight of
