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Visceral Leishmaniasis, or Kala Azar (KA): High Incidence of Refractoriness to Antimony Is Contributed by Anthroponotic Transmission via Post-KA Dermal Leishmaniasis Ruchi Singh,1 Dhiraj Kumar,1 Venkatesh Ramesh,2 Narender S. Negi,3 Surendra Singh,4 and Poonam Salotra1 1
Institute of Pathology, Indian Council of Medical Research, Safdarjung Hospital Campus, and Departments of 2Dermatology and 3Medicine, Safdarjung Hospital, New Delhi, and 4School of Studies in Microbiology, Jiwaji University, Gwalior, India
Visceral leishmaniasis, or kala azar (KA), is a chronic infection caused by the protozoan parasite Leishmania donovani, which is associated with high morbidity and mortality. Post–KA dermal leishmaniasis (PKDL) develops in 5%–15% of patients in India who are treated for KA. Cases of PKDL are of considerable epidemiological importance, acting as a reservoir of L. donovani Received 30 November 2005; accepted 27 February 2006; electronically published 22 June 2006. Potential conflicts of interest: none reported. Financial support: Indian Council of Medical Research (intramural funding to P.S.); Council of Scientific and Industrial Research (research fellowship to R.S. and D.K.). Reprints or correspondence: Dr. Poonam Salotra, Inst. of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi 110029, India (
[email protected]). The Journal of Infectious Diseases 2006; 194:302–6 2006 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2006/19403-0006$15.00
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Individuals with visceral leishmaniasis, or kala azar (KA) and individuals with post-KA dermal leishmaniasis (PKDL) are considered to be reservoirs of transmission of Leishmania donovani in India. When intracellular amastigotes were used to assess the natural susceptibility that PKDL isolates and KA isolates have to sodium antimony gluconate (SAG), the mean ED50 was found to be 12.0 2.49 and 11.0 1.38 mg/ mL, respectively; and there was a significant correlation with the clinical response (rrank p 0.99 ). All KA isolates, as well as a significant proportion (55%) of PKDL isolates from high-endemicity zones, were resistant to SAG. The median ED50 for SAG-resistant PKDL isolates (20.0 mg/mL) was significantly higher (P ! .05) than that for SAG-resistant KA isolates (15.7 mg/mL). SAG-resistant PKDL isolates may contribute to KA’s increased refractoriness to SAG, via anthroponotic transmission of SAG-resistant strains.
during interepidemic periods of KA [1]. The number of patients with KA who are unresponsive to the first-line drug sodium antimony gluconate (SAG) has reached alarming proportions (160%) in Bihar, India [2]. For a given dosage of SAG, effective treatment of PKDL cases requires parenteral SAG treatment for a period 14 times greater than that required for treatment of KA [3]. There is evidence that an inherent lack of susceptibility and/or acquired resistance by L. donovani contributes to KA’s unresponsiveness to the antimonials; for PKDL, however, such evidence is lacking [4–6]. The monitoring of antimonial resistance is a crucial issue in the anthroponotic focal region of leishmaniasis endemicity in India [2, 7]. The higher-dosage requirement for effective treatment of PKDL, as well as the increasing number of treatment failures in India, led us to investigate the role that anthroponotic transmission plays in the spreading of drug-resistant L. donovani as a possible cause of unresponsiveness to SAG. With this objective in view, we studied patients with either KA or PKDL who were from regions (e.g., Bihar and West Bengal) with zones of varying leishmaniasis endemicity, to determine L. donovani’s susceptibility to SAG. Also, to explore the possibility of development of markers for the monitoring of drug resistance, we sought to study both (1) the amplification of genes that, in vitro, have been shown to be involved in conferring resistance to leishmaniasis—that is, the genes pgpA, ldmdr, and gsh-1— and (2) the correlation between drug resistance and the efflux of rhodamine 123 (Rh123). Patients, materials, and methods. During 2001–2004, L. donovani isolates from bone-marrow aspirates from patients with KA (n p 19) and from dermal lesions from patients with PKDL (n p 18) were prepared, as described elsewhere [8]. Informed consent based on the guidelines of the Ethical Committee, Safdarjung Hospital, New Delhi, was obtained from all patients. The patients were from regions (e.g., Bihar and West Bengal) with zones of low, moderate, and high leishmaniasis endemicity, which represent, respectively, zones of low resistance (LR), moderate resistance (MR), and high resistance (HR) to antimony [2]. All the isolates, whether of KA or PKDL origin, were characterized as L. donovani, on the basis of species-specific monoclonal antibody and polymerase chain reaction [8]. Susceptibility to SAG was evaluated after isolation from patients and was based on the use of stationary-stage promastigotes, to mimic the host milieu. An intracellular assay for L. donovani was performed as described elsewhere [6]. Mouse-macrophage–adherent cell line J774A.1 (2 ⫻ 10 5 cells/well) in 8-well chamber slides (Nunc)
Table 1. Clinical profile of patients with kala azar (KA) who are from different zones of endemicity, and in vitro susceptibility of intracellular amastigotes to sodium antimony gluconate (SAG). Patient (age, years; sex)
ED50 (mg/mL) for amastigotesc
Region, zonea
Responseb
West Bengal, LR West Bengal, LR Munger, LR Munger, LR
R R ND R
2.1 3.4 3.9 4.7
0.28 0.28 0.28 0.42
Bhagalpur, LR Gopalganj, MR Araria, MR Siwan, MR
NR R R R
10.4 4.2 4.2 5.6
2.17 0.38 1.03 0.57
K9 (22; F) K10 (11; M)
Madhubani, HR Saran, HR
R ND
11.3 0.89 11.8 1.28
K11 (21; F) K12 (7; F)
Vaishali, HR Madhubani, HR
NR NRd
d
14.7 3.29 15.8 1.03
K13 K14 K15 K16
Saharsa, HR Saran, HR Darbhanga, HR Saran, HR
NR e … …e …e
19.4 1.68 13.0 4.12
K17 (17; F) K18 (23; F)
Madhubani, HR Saharsa, HR
… …e
e
17.3 4.32
K19 (24; M)
Saran, HR
…
e
20.3 0.84
K1 K2 K3 K4
(14; (20; (24; (10;
M) M) F) F)
K5 K6 K7 K8
40/ F (45; M) 7/M (36; F)
13.6 2.51 15.7 4.01 17.8 2.28
NOTE. HR, high resistance to SAG; LR, low resistance to SAG; MR, moderate resistance to SAG; ND, not determined; NR, not responsive to SAG; R, responsive to SAG. a
Based on data reported by Sundar [2]. After 30 days of treatment with SAG at a daily dose of 20 mg/kg body wt. Patients without fever and with reduction in spleen size were classified as R; the other patients were classified as NR. All patients were monitored for 6 months, and, if there were no signs of relapse, were considered to have been cured; during this period, none of the cured patients with KA developed post-KA dermal leishmaniasis. c For 3 assays. d Patient died during treatment. e Patient was treated with amphotericin B; therefore, responsiveness to SAG could not be determined. b
was infected with stationary-stage promastigotes at a 10:1 (L. donovani:macrophage) ratio and was incubated in 5% CO2 for 4 h at 37C. Unattached promastigotes were washed off, and the cells were incubated for 8–12 h. Infected cells were reincubated, for 48 h, with SAG concentrations of 0, 3, 10, 30, 60, and 100 mg/mL. After staining with Diff-Quick solutions, the number of L. donovani amastigotes per cell were counted, at ⫻100 magnification, in 100 macrophages. The percent killing was calculated by use of sigmoidal regression analysis (Origin 6.0; OriginLab), and both the ED50 and the ED90 were determined. Promastigotes at log phase were incubated in M-199 medium, with 5 mg/mL of Rh123 (Molecular Probes), for 45 min at 25C and were washed with PBS [9]. A total of 10,000 events were used in the analysis. Cell acquisition and analysis were performed by use of a FACS Calibur flow cytometer (Becton Dickinson) equipped with Cell Quest Software (Becton Dickinson).
A 5-mg portion of genomic DNA, isolated from L. donovani cultures by use of the Wizard Genomic DNA isolation kit (Promega), was digested, for 16 h at 37C, with restriction enzyme MvaI [8]. Southern blot analysis was performed with ldmdr (a multidrug-resistance gene), pgpA (an ABC transporter gene), and gsh1 (the gene for g-glutamylcysteine synthetase), which were labeled with 32P for use as probes [8, 10]. The Mann-Whitney U test was used to determine the statistical significance of the median ED50 values obtained. Spearman’s rank correlation coefficient was calculated to determine the correlation between the leishmaniasis endemicity of a zone, in vitro susceptibility, and the clinical response of patients. Results. The profiles of the patients with KA, as well as the KA isolates’ range of susceptibility to SAG, are summarized in table 1. On the basis of Thakur et al.’s [11] recent comprehensive study of the pharmacokinetics of SAG in Indian patients with KA, we categorized L. donovani as being resistant to SAG if the ED50 for SAG was 111.0 mg/mL. The mean SE BRIEF REPORT • JID 2006:194 (1 August) • 303
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(22; M) (12; M) (7; F) (20; M)
Table 2. Clinical profile of patients with post–kala azar dermal leishmaniasis (PKDL) who are from different zones of endemicity, and in vitro susceptibility of intracellular amastigotes to sodium antimony gluconate (SAG). Patient (age, years; sex)
Region, zonea
History of kala azar, years
Responseb
ED50 (mg/mL) for amastigotesc
ND R R R
4.8 2.9 4.2 6.3
0.23 0.13 0.56 0.24
Siwan, MR Araria, MR Gopalganj, MR Samastipur, HR
1 6 9 3
R SR ND R
6.4 8.5 36.0 3.0
0.33 0.78 3.35 0.41
P9 (12; M) P10 (20; M)
Darbhanga, HR Darbhanga, HR
4 0.5
R R
3.9 0.42 4.3 0.32
P11 (10; M) P12 (10; M)
Vaishali, HR Vaishali, HR
4 4
R R
4.7 1.04 5.5 1.19
P13 P14 P15 P16
Darbhanga, HR Vaishali, HR Saharsa, HR Darbhanga, HR
3 3 5 3
SR ND SR SR
7 No history
Relapsed ND
(18; (28; (20; (18;
M) M) M) M)
West Bengal, LR Siwan, MR Munger, LR Gopalganj, MR
P5 P6 P7 P8
(25; (25; (35; (32;
M) M) M) M)
(8; M) (40; M) (17; M) (23; M)
P17 (18; F) P18 (15; M)
Muzzafarpur, HR Madhubani, HR
12.4 15.3 17.9 20.0
2.03 2.98 3.32 2.64
28.7 1.02 31.2 4.5
NOTE. HR, high resistance to SAG; LR, low resistance to SAG; MR, moderate resistance to SAG; ND, not determined; R, responsive to SAG; SR, slow response to SAG. a
Based on data reported by Sundar [2]. All 18 patients were advised to take SAG, at a daily dose of either 10 mL (1000 mg), for patients ⭓12 years old, or 5 mL (500 mg), for patients !12 years old. On the basis of a previous study, patients who showed improvements ⭐4 weeks after treatment were classified as R; those who did so after this period were classified as SR [3]. All patients were monitored for 1 year, to assess improvement (e.g., such as a decrease in the size of papules and nodules and/or a regression of indurated areas) after treatment; because repigmentation is a slow process, improvement in those with a predominantly hypochromic macular presentation could be ascertained only toward the end of or after completion of the 4-month treatment period. c For 3 assays. b
ED50 for KA isolates (n p 19) as amastigotes was 11.0 1.38 mg/mL; for KA isolates from LR or MR zones, the corresponding value was 2.1–10.4 mg/mL. The endemicity zones of the KA isolates were strongly correlated with in vitro susceptibility (rrank p 0.997) and with the clinical response (rrank p 0.991). For 11 (58%) of the 19 KA isolates, the ED50 was 111.0 mg/mL; all 11 of these isolates were from an HR zone. The median ED50 for the SAG-resistant KA isolates was 15.7 mg/mL. The mean ED90 for SAG-susceptible KA isolates was 20.0–60.0 mg/mL, whereas that for SAG-resistant KA isolates was 1100.0 mg/mL. The characteristics of the patients with PKDL, as well as the PKDL isolates’ susceptibility to SAG, are summarized in table 2. The mean SE ED50 for PKDL isolates (n p 18) as amastigotes was 12.0 2.49 mg/mL. The ED50 for 6 (86%) of the 7 PKDL isolates from LR or MR zones was 2.9–8.5 mg/mL, indicating high susceptibility to SAG, whereas that for the remaining isolate was an exceptionally high 36.0 mg/mL. The ED50 for isolates from HR zones was 3.0–31.2 mg/mL, indicating varying degrees of susceptibility to SAG. The ED50 was 111.0 mg/mL for only 7 (39%) of the 18 PKDL isolates, and all but 304 • JID 2006:194 (1 August) • BRIEF REPORT
1 of these 7 were from an HR zone. The median ED50 for the SAG-resistant PKDL isolates was 20.0 mg/mL. The endemicity zones of the PKDL isolates were strongly correlated with in vitro susceptibility (rrank p 0.97) and with the clinical response (rrank p 0.95). The ED90 for SAG-susceptible PKDL isolates was 10.0–60.0 mg/mL, whereas that for SAG-resistant PKDL isolates was 1100.0 mg/mL. Overall, there was no significant difference between the mean ED50 (and ED90) for KA isolates and that for PKDL isolates, suggesting that the pharmacokinetics of SAG may not be responsible for the longer treatment required by patients with PKDL. When the susceptibilities of all the SAG-resistant isolates were compared, the median ED50 for the PKDL isolates was significantly higher (P ! .05 ) than that for the KA isolates, emphasizing the fact that prolonged exposure to SAG promotes L. donovani’s resistance to it. Because Rh123 has been found to be useful in the investigation of multidrug resistance in cancer-cell lines, the natural ability of KA isolates and PKDL isolates to accumulate Rh123 dye was studied. The median fluorescence intensity, which was
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15 8 8 3
P1 P2 P3 P4
(11.02 mg/mL) cases of KA in India are similar [11]. In the present study, there was no marked difference between the susceptibility of KA isolates and that of PKDL isolates, which suggests that the higher doses of SAG that are required for treatment of patients with PKDL may be necessitated by host factors. Studies of L. donovani in mice have demonstrated that a Th1 response is necessary for this parasite to be cleared by treatment with pentavalent antimony, suggesting that the in vivo efficacy of such treatment is at least partially based on immune response [12]. The development of PKDL has been associated with both increased production of interleukin (IL)– 10 by keratinocytes and high levels of IL-10 in plasma and in cultures of peripheral-blood mononuclear cells [13]. The longer treatment period required by PKDL may be attributable to a persistently high level of IL-10 during therapy, which has been associated both with poor response to treatment in patients with localized cutaneous leishmaniasis and with persistence of leishmaniasis in patients with L. mexicana infection [14]. The use of promastigotes to assess susceptibility to SAG and to trivalent antimony (data not shown) did not reveal any correlation between such susceptibility and the clinical response, a result that also had been reported in an earlier study [5]. The gene-amplification events associated with SAG-resistant L. donovani selected in vitro were not detectable in the clinical isolates, a finding similar to those of earlier reports [10, 15]. Intracellular amastigotes’ natural resistance to antimony is a multifactorial event, as indicated by altered levels of g-glutamylcysteine synthetase and ornithine decarboxylase, which are involved in thiol metabolism, and by reduced expression of aquaglyceroporin 1, which leads to decreased uptake of trivalent antimony [15]. The present study observed pgpA-gene polymorphic differences between the PKDL isolates and the KA isolates, which may indicate that, during persistence in the host after KA has been cured, L. donovani undergoes some genetic changes; previous studies have shown that such polymorphic differences exist in the 28S rRNA gene of L. donovani [8]. Rh123 efflux, which has been associated with the activity of efflux pumps such as pgpA and which has been used as an indicator of drug resistance, failed to provide a marker that could be used to monitor L. donovani’s drug resistance; in the absence of molecular markers for resistance to antimony, effective monitoring of drug resistance is possible via the technically demanding amastigote-macrophage model. Current knowledge of the epidemiological characteristics and transmission of leishmaniasis suggests that the spread of acquired drug resistance is a factor of major concern in focal regions of anthroponotic KA—especially in India, where individuals with PKDL serve as a major reservoir. The correlation between the preponderance of resistance to SAG, in the patients with PKDL, and in vitro susceptibility to L. donovani implicates patients with PKDL as being a source of L. donovani that is BRIEF REPORT • JID 2006:194 (1 August) • 305
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used to determine the extent of accumulation of Rh123, varied widely, from 552 to 1498, with the mean SD fluorescence intensities of the KA isolates and PKDL isolates being 828.12 257.33 and 859.33 364.55, respectively; however, there was no correlation between such fluorescence and drug resistance. Amplification of the ldmdr, gsh1, and pgpA genes was investigated by use of Southern blot analysis. Radiography showed similar band intensities in all KA isolates and PKDL isolates, for all the probes tested. Furthermore, polymorphisms in both the KA isolates and the PKDL isolates were observed at the pgpA locus, as indicated by an ∼1-kb polymorphic band, which was either absent or present at weak intensity in PKDL isolates, and by a 3-kb major band, which was present in all isolates. In the absence of amplification of the transporter molecule, accumulation of Rh123 cannot be correlated with L. donovani’s resistance to SAG, indicating that the SAG-resistant mechanism in clinical isolates is different from that in laboratory-selected SAG-resistant isolates. Discussion. Resistance to antimonials has emerged as a major barrier to the treatment and control of leishmaniasis in India. In northern Bihar, the magnitude of resistance varies from moderate to high, whereas patients from the southern districts of Bihar (i.e., south of the Ganges River) and from West Bengal respond well to treatment [2]. The present study considered L. donovani isolates from Indian patients with either KA or PKDL who were from zones of varying leishmaniasis endemicity and who had different responses to treatment with SAG. The results indicate that refractoriness to SAG is prevalent in KA isolates from HR zones, whereas a large majority of KA isolates from MR and LR zones are susceptible to SAG in vitro. In PKDL isolates from HR zones, in vitro responses were mixed, with both SAG-resistant and SAG-susceptible isolates being present. Because PKDL develops long after recovery from KA, the results of the present study do not rule out the possibility that patients with PKDL migrate from LR and MR zones to HR zones, and vice versa. In a majority of isolates, the response to SAG was similar to the clinical response to treatment. The present study revealed that 58% of KA isolates and 39% of PKDL isolates were resistant to SAG. L. donovani’s susceptibility is reported to decrease after 1 or more courses of treatment with SAG [4], and therefore the present study compared the KA isolates’ and the PKDL isolates’ susceptibility to SAG. Although the overall susceptibility to SAG was similar in the PKDL isolates and the KA isolates, the median ED50 for the SAG-resistant PKDL isolates was significantly higher than that for the SAG-resistant KA isolates. The intrinsic resistance of L. donovani to SAG may be increasing because of this parasite’s prolonged exposure to SAG during treatment of PKDL, as well as because of patients’ low compliance with the treatment regimen. It has been reported elsewhere that plasma levels of antimony in SAG-resistant (11.5 mg/mL) and SAG-susceptible
refractory to treatment with SAG and that may efficiently circulate in the community as SAG-susceptible L. donovani is eliminated by the drug and as the proportion of patients with L. donovani refractory to SAG rises.
7. 8.
Acknowledgments 9. We are grateful to Marc Ouellette (Faculte de Medecine, Universite Laval, Quebec, Canada) for the generous gift of pgpA and gsh-1 probes and to Buddy Ullman (Oregon Health and Science University, Portland) for the ldmdr1 probe. 10.
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