Indian Journal of Expe rimental Biology Vol. 39, March 2001, pp. 249-254

Studies on stibanate resistant Leishmania donovani isolates of Indian origin

S Pal , A Mandai & S Duttagupta Leishmania Divi sion,lndian Institute of Chemical Biology, 4, Raja S.c. Mullick Road, Calcutta 700032 , Indi a

Received 5 April 1999; revised 9 November 2000

Studies with 26 clones of L. dOllovalli promasti gotes derived from three different Indian isolates indicated that wild type parasites are mixture of stibanate sensitive and resistant cells. Both forms of the parasite were resistant to the drug. Infection with resistant parasites appears to be the primary reason of high rate of pentavalent antimony unresponsiveness among Indian kala-azar patients. It was observed that the resistant parasites originated as a result of irregular and often incomplete treatment of kala-azar patients with pentavalent antimonials.

Parasitic protozoa belonging to the genus Leishmania are the causative agents of a wide spectrum of diseases collectively known as leishmaniases . Visceral leishmaniasis (VL) or kala-azar is generally caused by members of Leishmania dOllovani complex, namely, L. r/onovani, L. infantum and L. chagasi. The disease is potentially fatal to humans and if remains untreated, mortality rate is over 95%. Organic pentavalent antimonials [Sb(V)] in the form of sodium stibogluconate (pentostam, solustibosan, stibanate) or meglumine antimonate (glucantime) are the drugs of first choice for treatment of VL and all other forms of leishmaniasis I . When treatment with Sb(V) fails , pentamidine or amphotericin B are used as drugs of second choice2

.3. Many and frequent side effects are the causes of their limited use. Allopurinals have also been used successfully for treatment of antimony unresponsive VL patients in Kenya and Bihar4

.5. Drug resistance is a major barrier in the treatment and control of parasitic diseases. Unresponsiveness to antimony chemotherapy in VL and mucocutaneous leishmaniasis in particular has

long been recognised as a serious clinical problem6•7

•

Increasing number of reports8•

11 about initial failure to Sb(V) therapy or relapse after apparently successful prior treatment is the basis of this conclusion.

Objective of the present study was to generate Sb(V) resistant parasites from wild type natural isolates and to examine whether development of resistance in parasites is one of the main reason for this alarming rise in Sb(V) unresponsiveness among Indian kala-azar patients.

Address for correspondence: Sampa Pal, Seatt le Biomedical Research Institute, 4, Nickerson Street, Suite 200, Seattle, WA98 109

Materials and Methods Cell cuLture-L. dOllovalli promastigotes were

routinely maintained in M-199 (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (lnnovar Biological Incorporated, USA) containing 25 mM Hepes (Life Technologies, USA, pH 7.5), streptomycin (100 )lg/mL) and penicillin (100 units/mL; Sigma Chemical Company, USA). Parasites were subcultured at 5-7 days interval at an initial cell density of 2.5 x 105/mL. L. dOllovalli isolates used in this study were, MHOM/IN/83/AG83 (AG83), MHOM/IN/88/GEI (GEl), (MHOM/IN/881 GE2 (GE2), MHOM/IN/94/RS (RS), MHOM/IN/951 MF (MF) and (MHOMIIN/95/CK (CK). AG83, GEl and GE2 were isolated from kala-azar patients responsive to Sb(V) therapy, whereas, RS was isolated from a post kala-azar dermal leishmaniasis (PKDL) patient who had kala-azar in 1991 , treated with Stibanate and was completely cured. From 1992 onwards the patient showed typical symptoms of PKDL with progressively increasing depigmented patches along with appearance of nodules in face , neck and abdomen. Parasites were isolated from foreskin scrapings of right ear lobule before any treatment. MF and CK were isolated from bone marrow aspirates of Sb(V) unresponsive kala-azar patients.

DeveLopment of stiballate Ullre5pOllsive parasites ­Stibanate (sodium antimony g luconate) resistant promastigotes of AG83, GEl and GE2 isolates were developed in four steps by ill vitro passage of parasites (5 x 105/mL) in presence of 0.5, I, 2 and 3 mg/ml of stibanate (Gluconate Health Limited, India) in the medium. In each step, parasi tes were cultured for at leas t 5-7 passages to attain steady and

250 INDI AN J EXP BIOL, MARCH 200 1

optimal ce ll growth . To generate resistant paras ites ill vivo, Syrian golden hamsters were intracardially inj ected with suspension (0.1 mL) of wild type AG83

promastigotes (I x 107) and after 7 days the animal s

were subcutaneously injected with suboptimal dose ( IOOmg/kg body wt/day) of stibanate for two consecutive days as described by Trotter et al. 12

• On day 7 after stibanate treatment, the animals were sacrificed and isolated spleen cells were cultured at 22°-24°C in liquid medium in presence of different concentrations of stibanate for isolation of stibanate resistant parasites.

Cloning -Promastigotes of different isolates were cloned by limited dilution. In short, exponentially growing cells were serially diluted to less than 5 cells/mL in M-199 supplemented with 20% of fetal bovine serum (FBS) and 50% of conditioned medium. of cell suspension (100 )..lL) was added in 96 wells bored in culture plates (Costar, USA) and incubated at 22°-24°C for 2-3 weeks. Any plate having more than 6 positive wells were discarded. Filter sterili sed culture supernatant from exponentially growing AG83 promastigotes in M-199 containing FBS (20%) was used as conditioned medium.

Drug sensitivity assay--To evaluate stibanate sensi ti vity ill vitro, promastigotes (5 x 105 cells/mL) were incubated at 22°_24° C in presence of various concentrations of the drug for 10-12 days and growth of the paras ites was monitored by cell counting. To test drug sensiti vity ill vivo, two groups of Syrian golden hamsters (12 animals in each group) were infected intracardially with stibanate sensitive and resistant clones derived from wild type GE 1. After seven days, six animals in each group were injected subcutaneously with complete dose (400 mg/kg/day) of stibanate for five consecutive days as described by Trotter et al. 12 and animals were monitored for 6 months.

Results Two stibanate resistant cell lines were generated

from wild type sensitive AG83 promastigotes that could survive and multiply in presence of 3 mg/mL of stibanate. Stibanate resistant cell line, AG83R I was developed by repeated in vitro passages of promastigotes in presence of increasing concentration of stibanate; and another resistant cell line AG83R2 was isolated from ill vitro culture of spleen cells of infected hamsters treated with suboptimal doses of stibanate. Results presented in Table I indicated that in absence of stibanate, wi ld type promastigotes of

AG83, GEl and GE2 mult iply nearly 30-45 times in 8 days while more than 90% of the same promastigotes even failed to survive in presence of 3mg/mL of the drug. Therefore, it appeared that st ibanate had lethal effec t on ill vitro growth of three different wild type natural isolates of L. donovani promastigotes. Under identical conditions, the drug had no lethal effect on resistant promastigotes, namely, AG83RI or AG83R2 (Table I) . Similarly , stibanate had no lethal effect on three other wild type promas ti gotes, namely , CK and MF isolated from two Sb(V) unresponsive kala-azar patients and RS isolated from a PKDL patient. It was observed that all resistant paras ites, namely, AG83R I, AG83R2, CK, MF and RS multiplied nearly J 5 to 35 times in presence of 3 mg/mL of stibanate and growth of all res istan t parasites except RS was partially inhibited in presence of the drug (Table I). Growth patterns of AG83, AG83R I and RS representing one each of a sensitive, a res istant and a PKDL isolate in presence and absence of 3 mg/ml of stibanate over a period of 12 days have been shown in Figure 1. Growth patterns of other sensiti ve (GEl and GE2) and resistant isolates (AG83R2, CK and MF) were similar to that of AG83 and AG83 RI respect ive ly (data not shown).

Over 90% of wild type AG83 promastigotes died in presence of stibanate (3 mg/ml), but some parasi tes remained viable even after two weeks. Unlike normal promastigotes, morphologically these viable paras ites

Table I-Effect of stibanate on ill vitro growth of different L. dOllovalli promastigotes

rValues are mean ± SO of 3 independent experiments]

Isolate Stibanate Cell coun t (mg/mL) x 10-6

AG83 0 14.6±1.2 -do- 3 <0.05 GEl 0 19.0± 1.2 -dO- 3 <0.05 GE2 0 22.0± 1.7 -do- 3 <0.05 AGS3 RI 0 11.6±1.7 -do- 3 7.9±0.1 AGS3R2 0 25.0±2.4 -do- 3 IS.0± 1.6 CK 0 16.0± 1.2 -do- 3 II.O ±O.S MF 0 2 1.6 ± 1.7 -dO- 3 16.0±2.9 RS 0 17.0±2. 1 -dO- 3 17.0± 1.6

"Init ial cell counts were I x 106/mL in case of AGS3R2 and 5 x 105/mL for al l other isolates.

I

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PAL et al.: STUDIES ON STIBANATE RESISTANT LEISHMANIA DONOVANIISOLATES OF INDIAN ORIGIN 251

were oval shape with a very short flagellum and could hardly move. Ability of few promastigotes to survive in presence of stibanate raised the possibility that wild type natural isolates of Leishmania were the mixed population of cells having varying degree of sensitivity towards the drug. To examine whether wild type isolates of Leishmania were the mixture of stibanate sensitive and resistant cells, AG83, GEl and GE2 promastigotes were cloned by limited dilution. Out of total 26 clones, 13 clones were obtained from AG83, 3 from GEl and the rest were from GE2 respectively. Stibanate sensitivity assay indicated that nearly 33% of the clones of both AG83 and GEl, and 80% of the clones of GE2 were resistant to 3 mg/mL of stibanate in culture (Table 2). Therefore, it appeared that wild type natural isolates of Leishmania parasites of Indian origin isolated from kala-azar patients responsive to Sb(Y) therapy were the mixture of sensitive and resistant cells. In contrast, all clones derived from CK, MF, RS and AG83R 1 were stibanate resistant indicating that unlike AG83, GEl or GE2, wild type promastigotes CK, MF , RS and AG83R I consisted of a homogeneous population of stibanate resistant cells.

To test whether the resistant promastigotes also remained resistant after being transformed to amastigotes in vivo, hamsters were infected with two clones derived from GEI-stibanate sensItIve (GEIC6S) and stibanate resistant (GEIF8R). In vitro growth of these clones has been shown in Fig. 2. In vitro growth of other sensitive and resistant clones

Table 2-11/ vitro stibanate sensitivity of various clones of L. dOl/oval/i promasti gotes

Isolate Number of clones Total Sensitive (%) Resistant (%)

AG83 13 9 (69) 4 (31) GEl 3 2 (67) I (33) GE2 10 2 (20) 8 (80) AG83RI 7 o (nil) 7 (100) CK 3 o (nil) 3 (100) MF 4 o (nil) 4 ( 100) RS 4 o (nil ) 4 (100)

All conditions are same as in Table I except that initial cell counts were 5 x 10'/mL for all isolates.

Table :'- In vivo stibanate response of two clones L. dOl/ovani promastigotes

Isolate Treatment Survival (%)

GEIC6S None 0/6 (nil) -do- Stibanate 4/6 (67) GEIF8R None 1/6 (17) -do- Stibanate 116 (17)

were similar to that of GEIC6S and GEl F8R respectively (data not shown). Results presented in Table 3 indicated that 5 out of 6 hamsters infected with the sensitive clone, GEIC6S, survived following treatment with stibanate, whereas, under identical conditions only lout of 6 hamsters survived after

180 .,-----------------,

160

140

6 120

x 100 C ::J

8 80 Q)

U 60

40

20

2 4 6 8 10 12

Day

Fi g. I-Effect of stibanate (3 mg/mLO on iI/ vitro growth of wild type L. dOl/ovani promastigotes. A- AG83 in presence of drug; B-AG83 in absence of drug ; C-AG83RI in presence of drug; D-AG83RI in absence of drug; E-RS in presence of drug; and F-RS in absence of drug. Results shown here represen t one of at least four experiments, each of which gave similar results . Other conditions are same as in Table I.

180 r--------------------------~

160

140 III

d 120

x 100 C

::J 80 0

u

Q) 60 U

40

20

o 0 2 4 6 8 10 12 Day

Fig. 2-Effect of st ilbanate (3 mg/mLO on in vitro growth of two clones of L. dOl/oval/i promastigotes. A-GEIC6S in presence of drug; B -GE I C6S in absence of drug; C-GE I F8R in presence of drug; and D-GE I F8R in absence of drug. Other conditi ons are same as in Table I.

252 INDIAN J EXP BIOL, MARCH 2001

infection with the resistant clone, GE I F8R. Therefore, these results indicated that ill vitro stibanate sensitivity or resistance of parasites did not change following transformation within experimental hosts. As expected, without stibanate treatment, over 80% of hamsters died within 2-3 months irrespective of whether they were infected with the sensitive or resistant clone.

Discussion Cases of VL as well as other forms of

leishmaniases refractory to Sb(V) therapy are known for a long time in many endemic areas including

India 13• However, the exact correlation between

Sb(V) resistance and treatment failure still remains obscure. In earlier studies l4.15 clonal population of parasites have been used to generate Sb(V) resistant promastigotes. We have used wild type natural isolates of L. dOllovoni for development of Sb(V) resistant mutants as infection of naive hosts occurs with wild type parasites. Development of Sb(V) resistance was a slow process and was achieved only when the parasites in culture were exposed to step wise increase in the drug concentrations for nearly 30-40 passages. All resistant promastigotes irrespective of their origin grew nearly 15-35 times in 7 days in presence of 3 mg/mL of the drug. Attempts to generate promastigotes that can grow in more than 3 mg/mL of stibanate was not successful. Using a Sudanese isolate of L. donovoni promastigotes, Ullman et 01.16 have generated two cell lines PENT 0400 and PENT 03200 that are resistant to 0.4 and 3.2 mg/mL of pentostam respectively. In contrast, resistant promastigotes generated from patients with American cutaneous leishmaniasis can grow in presence of 3-4 fold higher concentrations of Sb(V)1 4. 15. The degree of resistance exhibited by L. dOllovoni promastigotes of Indian origin was

similar to that of Sudanese isolate of L. donovolli l6•

We have observed that compared to wild type parasites, the resistant cells grew slowly and attained lower cell density in presence of the drug. However, when they were grown in absence of the drug for 2-3 passages, they remained resistant to Sb(V) but growth rate and optimal cell density became similar to that of wild type cells. Therefore, slower growth rate of the resistant parasites in pre ence of stibanate was probably due to non-specific inhibitory effect of the drug. In vitro growth of PENT 0400 and PENT 03200 has also been found to be partially inhibited in presence of pentostaml 6

. Our result was also in

agreement with Ullman's findings where they have reported that pentostam resistance of PENT0400 and PENT03200 is a stable genetic trait. Analysis of stibanate sensitivity of various clones derived from 3 different isolates indicated that 13 out of 26 were resistant to the drug ill vitro. Our results demonstrated that natural wild type L. donovolli promastigotes of Indian origin were a mixture of stibanate sensitive and resistant parasites . Grogl et 01.17 have also observed that wild type promastigotes of Leishmania spp. isolated from patients with American cutaneous leishmaniasis represent a heterogeneous population as demonstrated by a biphasic concentration response to Sb(V) which is typical of mixed population . It appeared that development of Sb(V) resistance in the present case was due to selection of resistant cells' from the mixed population under continuous drug pressure. This conclusion was also supported by the fact that all clones derived from wild type AG83R I, the resistant cell line developed in vitro were resistant to 3 mg/mL stibanate. Our results also showed that the proportion of Sb(V) resistant parasites present in wild type GEl was nearly same in both AG83 and GEl but was significantly higher in GE2. Apparent reason(s) for this difference was not clear as all three were isolated from kala-azar patients responsive to Sb(V) treatment. It might be possible that GE2 was isolated from a patient who responded to treatment with higher doses of Sb(V) or for a longer duration or combination of both . Whether these arguments were valid or not could not be assertained as detail clinical history of the patient was not available.

Recently Ibrahim et 01.1 8 have demonstrated that a mutant of L. major promastigote is resistant to I mg/mL of pentosam whereas amastigotes are sensitive to as low as 20 and 40 !lg/mL of the drug. Using two clones of GEl, we have clearly demonstrated that L. dOllovani promastigotes and amastigotes were equally affected by the particular concentration of stibanate. Previously drug sensitivity of amastigotes was tested in vitro using isolated macrophage system 19.20. This is probably the first report using experimental animals that Sb(V) resistant promastigotes are also resistant to the drug in vivo. Our results also suggested that steady increase in number of kala-azar patients unresponsive to treatment with Sb(V) was mainly due to infection with truly Sb(V) resistant parasites. This conclusion was strongly supported by the fact that all clones derived from CK and MF isolated from two Sb(V) unresponsive kala-azar patients were resistant to 3

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PAL et al. : STUDIES ON STIBANATE RESISTANT LEISHMANIA DONOVANIISOLATES OF INDI AN ORIGIN 253

mg/mL of stibanate. Since resistant parasites could be eas ily isolated fro m infected hamsters treated with incomplete doses of stibanate, irregular and incomplete treatment appeared to be the principle reason of Sb(V) unresponsiveness. Steady ri se in number of Sb(V) unresponsive kala-azar cases was therefore, primarily due to infection with Sb(V) resistant parasites generated and selected out as a result of wide and at times abusive use of antimonial drugs.

Until recently nothing was known about the mechanism of Sb(V) res istance in Leishmania except that the res istant paras ites accumulate less radioactive pentostam than the wild type21 cells. It has been proposed that Sb (V) / As (V) containing compounds, including antileishmani al drug pentostam, are reduced intrace llularly to Sb (III) / As (III), conjugate to trypanothione, and extruded by As-thiol pump. In the present study, stibanate resistant promastigotes were found to be sensiti ve to both Sb (III) and As (III) [data not shown] . Similarly, pentostam resistant cell lines PENT0400 and PENT 03200 have also been found to be sensiti ve to growth inhibition and cytotoxicity caused by SbCI3 and SbCI5 as well as to a variety of other cations as Cd, Zn and As 16 . Therefore, mechanism(s) other than extrusion as thiol conjugate was operative in stibanate res istance in L. dOll 0 vall i. Amplification of P-glycoprotein related genes has also been observed in arsenite resistant cells22 where P-glycoprotei ns are involved in extrusion of hydrophobic drugs from mammalian cells23

. Further studies are needed to determine the exact mechanism of Sb(V) resistance in L. dOl/oval/i, a human pathogen.

Acknowledgement Financial support by OBI , New Delhi , Indi a, is

acknowledged .

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