Abstract.
Liposomal amphotericin B (LAmB) is recommended for treatment of Indian visceral leishmaniasis (VL), with a cure rate of more than 95% in the Indian subcontinent. A prospective observational study of 1,143 subjects was performed with a longer follow-up than prior studies (12 months) to evaluate the long-term effectiveness and safety of LAmB for the treatment of VL. Patients received a single dose of 10 mg/kg LAmB and were evaluated for initial cure at day 30 and final cure at 6 and 12 months to see the response to the therapy. Furthermore, predictors of relapse were also calculated. At day 30, the initial cure rate was 100%; however, at 6 months and 12 months, cure rates were 97.0% and 94.2% by per-protocol analysis and 96.9% and 93.9% by intension-to-treat analysis, respectively. Fever was the most common adverse event (AE). There were no deaths and serious AEs. Male gender, weight less than 30 kg, and spleen size more than 4 cm at the start of the treatment were significant risk factors of relapse. Liposomal amphotericin B was found to be very effective and safe in the treatment of VL. A longer follow-up period of 12 months is recommended to pick up late relapses.
INTRODUCTION
In 2005, governments of India, Nepal, and Bangladesh initiated a visceral leishmaniasis (VL) elimination program with the aim to reduce its incidence to less than 1 per 10,000 people by 2015. The target was then extended to 2017.1 In January 2012, the London Declaration on Neglected Tropical Diseases was launched, inspired by 2020 roadmap of the WHO, which aims to eradicate or prevent transmission of neglected tropical diseases including VL.2 Drugs for the treatment of VL are limited and some of them are associated with serious toxicities. Liposomal formulations of amphotericin B (AmB) have been an important breakthrough in the treatment of VL. The tolerance of AmB is greatly improved and adverse events (AEs), including nephrotoxicity, are minimized, and these enable the delivery of large doses of the drug over a shorter duration. Of all lipid formulations of AmB, liposomal amphotericin B (LAmB) (AmBisome; Gilead Sciences, Foster City, CA) has been extensively used for the treatment of VL with excellent results.3–8 A single dose of 10 mg/kg LAmB achieved a cure rate of 95.7% in the Indian subcontinent, which led to a paradigm shift in the approach to the treatment of VL.8 After this trial, this single-dose regimen has been recommended as the most preferred regime for VL in South Asia by the WHO.9,10 The benefits of this treatment are excellent efficacy, assured compliance, and substantially reduced hospitalization cost and time, thus enabling treatment of a larger number of patients. Encouraged by the excellent efficacy at 6 months, an observational study was performed using a single dose of LAmB for the treatment of VL.
METHODOLOGY
Study design.
This prospective, observational study was carried out at the VL treatment site at the Kala-Azar Medical Research Centre (KAMRC); Muzaffarpur, Bihar, India, between June 2013 and February 2017 and was approved by the Ethics Committee of the KAMRC. The study was registered on clinical trials.gov (NCT01566552).
Written informed consent was obtained from patients and from guardians in case of children younger than 18 years. All authors participated fully in the design of the study, had access to all study data, and took responsibility for data analysis.
Study drug.
Liposomal amphotericin B (AmBisome; Gilead Sciences) was administered as a single dose of 10 mg/kg by intravenous infusion in 5% dextrose solution over 2 hours.
Study patients.
Patients of all ages and of both genders, having signs and symptoms suggestive of VL, that is, fever with chills and rigor for 2 weeks or more, not responding to antimalarial drugs, and splenomegaly were included in the study. An rK39 antigen–based immunochromatographic serological rapid test was performed. Patients with typical signs and symptoms with characteristic laboratory features (pancytopenia) and a positive strip test were included in this study. Patients with serious illnesses or concurrent infections, such as tuberculosis or bacterial pneumonia, HIV, and Hepatitis B/C, and known allergy to AmB were excluded.
Study protocol.
Patients were admitted for treatment at the KAMRC. Initial evaluation was performed by taking vital signs such as temperature, pulse, and blood pressure, and recording of other parameters such as weight and spleen size. Hematological and biochemical parameters were recorded at day 0, 1, and 30. The assessment of “initial cure” at day 30 consisted of a clinical examination, including temperature, weight, and liver and spleen size assessment, and evaluation of laboratory (hematological and biochemical) parameters. In patients with persistent signs and symptoms at day 30, splenic aspirate was performed and smears were examined for Leishmania parasites (LD bodies). Patients were instructed to visit the center if they experience recurrence of clinical symptoms of VL. If relapse was suspected on their visit, parasitological confirmation was performed by splenic smear examination. Relapse was defined as the recurrence of clinical symptoms of VL and the presence of parasites in splenic smear at follow-up.
Effectiveness.
“Initial cure” was defined as resolution of fever, regression of splenic enlargement, and recovery of laboratory parameters toward normal on day 30. All patients with initial cure were discharged from the clinic and invited for follow-up visits. “Final/definitive cure” was assessed at 6 and 12 months after the treatment. “Treatment failure” was defined as either the lack of initial cure at day 30 or relapse during follow-up. Patients with treatment failure or severe AEs of common terminology criteria (CTC) grade 3 or above were given rescue treatment with AmB (0.75 mg/kg administered for 15 daily infusions).11
Statistical analysis.
Data were extracted and were analyzed using statistical package for social sciences software (SPSS, Inc., Chicago, IL) for windows (Version 16.0). The data were presented as mean ± SD for continuous variables and as frequency with their respective percentages for categorical variables. Patient characteristics were described in terms of median with their interquartile range for skewed continuous data. For categorical data, chi-squared test was used, and for continuous data, Student’s t-test and Mann–Whitney U test were used. For paired samples, paired t-test and the Wilcoxon signed rank test were used. For the comparison of clinical outcomes in naïve versus those patients relapsing after prior antileishmanial treatment, independent t-test and Mann–Whitney U test were used. A multivariable analysis was performed by using the binary logistic regression model, including all variables that were associated with the outcome (P < 0.05) in the bivariable analysis. P-value of < 0.05 was considered statistically significant.
RESULT
A total of 1,143 patients were enrolled during the study period. After initial assessment, each patient was given the treatment. Demographic profile and clinical features of the patients are mentioned in Table 1. Fever was present in all patients with a mean temperature of 101.4 (SD: 7.37) degree Fahrenheit (°F) and for a median duration of 30 (IQR: 17–60) days. Forty-four patients had a history of previous treatment for VL, 39 with oral miltefosine and five with injection sodium stibogluconate.
Table 1.
Demographic profile of study population (n = 1,143)
| Characteristics | n = 1,143 |
|---|---|
| Age* (years) | 15 (8–32) |
| Gender, no. (%) | |
| Male | 689 (63.3%) |
| Female | 454 (39.7%) |
| Weight* (kg) | 30.0 (18.0–45.0) |
| District | 980 (85.7%) |
| Muzaffarpur | 160 (14.0%) |
| Other than Muzaffarpur | 3 (0.3%) |
| Uttar Pradesh | 0.00 |
| Previously treated for visceral leishmaniasis | 44 (3.8%) |
| Clinical features | |
| Fever | 1,143 (100%) |
| Generalized weakness | 56 (4.9%) |
| Rigor | 248 (21.7%) |
| Cough | 99 (8.7%) |
| Vomiting | 11 (1.0%) |
| Loss of appetite | 193 (16.9%) |
| Loss of weight | 46 (4.0%) |
| Spleen size* (cm) | 4 (2–6) |
| Liver size* (cm) | 2 (1–3) |
* Median (IQR = interquartile range).
Table 2 shows the various clinical, biochemical, and hematological characteristics of patients at day 0 (pretreatment) and 30. There was significant improvement on all parameters at day 30 when compared with the day of admission.
Table 2.
Comparison of clinical, hematological, and biochemical parameters at the start of the treatment and at day 30 of treatment
| Variable | Day 1 (n = 1,143) | Day 30 (n = 1,088)* | Day 1 vs. Day 30 (P value) |
|---|---|---|---|
| Creatinine† (mg/dL) | 0.69 (0.26) | 0.63 (0.25) | < 0.001 |
| Serum glutamic pyruvic transaminase‡ (IU) | 37 (24–62) | 32 (23–43) | < 0.001 |
| Hemoglobin‡ (gm/dL) | 8.0 (7.0–9.50) | 11.03 (9.4–11.11) | < 0.001 |
| Platelet count† (/mm3) | 101,000.00 (53,218.26) | 254,000.00 (95,234.42) | < 0.001 |
| White blood cell count† (/mm3) | 4,393.26 (2,074.49) | 10,700.00 (5,729.53) | < 0.001 |
| Spleen‡ Size (cm) | 4 (2–6) | 2 (2–3) | < 0.001 |
| Liver size‡ (cm) | 2 (1–3) | 2 (1–2) | < 0.001 |
| Weight‡ (kg) | 33 (18–35) | 35 (20–46) | < 0.001 |
| Fever (°F) | 101.4 (7.37) | 97.5 (26.84) | < 0.001 |
* In two patients, drug was stopped because of hypersensitivity reaction and 53 patients were lost to follow-up at day 30.
† Mean (SD).
‡ Median (IQR = interquartile range).
Thirty-two patients (2.8%) experienced infusion-related chills. Rigor, vomiting, back pain, and diarrhea occurred in three, two, two, and one patients, respectively. However, these were CTC grade 1–2 and resolved without any complications.
Drug was discontinued in two patients who developed hypersensitivity reaction within 2 hours of infusion with LAmB but resolved spontaneously on cessation of drug infusion. At day 30, 53 (4.6%) patients were lost to follow-up. Remaining patients (n = 1,088; 95.2%) improved clinically, with significant improvement in weight and regression of spleen size (Table 2). However, 34 patients relapsed within 6 months and another 32 relapsed between 6 and 12 months. So, the cure rate at 6 and 12 months was found to be 97.0% and 94.2%, respectively, by per-protocol analysis. By intention-to-treat analysis, the cure rate was 96.9% at 6 months and 93.9% at 12 months.
Male gender, weight, and spleen size were significant risk factors for the predictors of relapse in bivariable analysis at 5% (i.e., P < 0.05). Those variables, significant at 5% (i.e., P < 0.05) in the bivariable model, were taken into the multivariable model. Males were twice (OR: 2.336, 95% CI: 1.303–4.187) more likely to suffer from relapse than females. Patients with weight ≤ 30 kg at day 0 were almost two times (OR = 1.947, 95% CI: 1.163–3.262) more prone to suffer from relapse than those with weight > 30 kg. Patients with spleen size > 4 at day 0 were two times (OR = 1.998, 95% CI: 1.204–3.316) more likely to suffer from relapse than subjects with spleen size ≤ 4. There was essentially no colinearity between the variables (gender, weight, and spleen size) that predicted relapse because the bivariate P-values were very close to the multivariate P-values (Table 3).
Table 3.
Factors associated with visceral leishmaniasis relapse after treatment
| Bivariable | Multivariable | |||||
|---|---|---|---|---|---|---|
| Characteristics | Total (1,143) N (%) | Non-relapse N (%) | Relapse (66) N (%) | P-value | OR (95% CI) | P-value |
| Age† (years) | ||||||
| ≤ 15 | 607 (53.10) | 565 (52.50) | 42 (63.60) | 0.08 | – | – |
| > 15 | 536 (46.90) | 512 (47.50) | 24 (36.40) | – | – | – |
| 15 (8.00–32.00)* | – | – | – | – | – | |
| Gender | ||||||
| Male | 689 (60.30) | 639 (59.30) | 50 (75.80) | 0.01† | 2.336 (1.30–4.18) | 0.00† |
| Female | 454 (39.70) | 438 (40.70) | 16 (24.20) | – | – | – |
| Weight† (kg) | ||||||
| ≤ 30 | 538 (47.10) | 498 (46.20) | 40 (60.60) | 0.02† | 1.94 (1.16–3.26) | 0.01† |
| > 30 | 605 (52.90) | 579 (53.80) | 26 (39.40) | – | – | – |
| 30.0 (18.00–45.00)* | – | – | – | – | – | |
| Hemoglobin† (gm/dL) | ||||||
| ≤ 8 | 540 (47.20) | 519 (48.20) | 38 (57.60) | 0.13 | – | – |
| > 8 | 603 (52.80) | 558 (51.80) | 28 (42.40) | – | – | – |
| 8.1 (6.90–9.50)* | – | – | – | – | – | |
| Spleen size† (cm) | ||||||
| ≤ 4 | 676 (59.10) | 648 (60.20) | 28 (42.40) | 0.00† | – | 0.00† |
| > 4 | 467 (40.90) | 429 (39.80) | 38 (57.60) | – | 1.998 (1.20–3.31) | – |
| 4.0 (2.0–6.0)* | – | – | – | – | – | |
* Median (IQR = interquartile range) of age, weight, hemoglobin, and spleen size.
† P < 0.05 Statistically Significant.
DISCUSSION
Liposomal amphotericin B has been shown to be very effective and safe among the basket of available antileishmanial drugs. A cure rate of > 97% at 6 months in our study was comparable with various studies of LAmB shown in Table 4 and in accordance with the recommendation of the WHO to judge efficacy.9 However, cure rates in most of the studies are calculated after 6 months of follow-up. We did extend the follow-up to 12 months and found that almost equal number of patients relapsed between 6 and 12 months (n = 32) as compared with patients who relapsed within 6 months (n = 34). Several studies have shown that relapses do occur after 6 months. Burza et al.,12 in a study found that when LAmB was given at doses of 5 mg/kg over 4–10 days to 8,749 patients with VL, 119 patients relapsed and of these, 18 (15.1%) patients relapsed within 6 months, 63 (52.9%) within 6–12 months, and 38 (31.9%) > 12 months after treatment. In another study by Burza et al.,13 relapses at 6-, 12-, and 15-month follow-up were 0.3% (2/767), 3.7% (14/383), and 2.4% (4/164), respectively. In another study from Nepal, in VL patients treated with miltefosine, the relapse rate at 6 and 12 months was 10.8% (95% CI: 5.2–16.4) and 20.0% (95% CI: 12.8–27.2; cumulative), respectively.14 Consistent with the finding of relapses even after 6 months of treatment, we would suggest that at least a 12-month follow-up should be used for various clinical trials and programs for early detection and treatment. Recently an open-label, prospective, nonrandomized, noncomparative, multicenter trial was conducted within public health facilities in two highly endemic districts and a specialist referral center in Bihar, India. Of 1,761 patients recruited, 50.6% (n = 891) received a single dose of LAmB. On monitoring for 6 months, in the intention to treat analysis, the final cure rate was 91.4% (95% CI: 89.3–93) for single dose of AmBisome. In the complete case analysis, the cure rate was 95.5% (95% CI: 93.9–96.8) for single dose of AmBisome.15
Table 4.
Various trials of treatment of visceral leishmaniasis with LAmB
| Serial no. | Author | Type of study | Dose used | Overall cure rate Percentage (6 month) |
|---|---|---|---|---|
| 1 | Thakur et al.3 | Randomized study | Group A: LAmB 15 mg/kg single-dose (i.v.) Group B: AmB at a dose of 1 mg/kg for 20 days.(i.v.) | 100% 100% |
| 2 | Sundar et al.4 | Randomized, dose-ranging, multicenter study | 0.75 mg/kg/days for 5 days (i.v.) vs. 1.5 mg/kg/days for 5 days (i.v.) vs. 3 mg/kg/days for 5 days (i.v.) | 89% vs. 93% vs. 96% |
| 3 | Sundar et al.7 | Open-label randomized comparative study | 5.0 mg/kg/days for 5 days (i.v.) | 90% |
| 4 | Sundar et al.6 | Open-label randomized controlled study | 10 mg/kg in a single infusion | 95.7% |
| 5 | Sundar et al.7 | Prospective, multicentric, randomized, open-label, comparative phase III study. | 3∶1 ratio to receive 15 mg/kg single infusion of either amphotericin B lipid emulsion (N = 376) or LAmB (N = 124) | 85.9% with ABLE 98.4% with LAmB |
| 6 | Sinha et al.8 | Observational cohort study | 20 mg/kg in four doses | 98.8% |
| 7 | Mondal et al.16 | Prospective open-label study | 10 mg/kg | 97% |
| 8 | Lucero et al.17 | Retrospective study | 15 mg/kg LAmB in three equal doses of 5 mg/kg | 98.7% (6 months) 96.4% (12 months) |
| 9 | Goyal et al.15 | Observational study | 10 mg/kg in a single infusion | 91.4% |
ABLE = amphotericin B lipid emulsion; LAmB = liposomal amphotericin B.
Declining efficacy of sodium stibogluconate and miltefosine and relapses occurring between 6 and 12 months of therapy with single-dose LAmB is a matter of concern and cannot be ignored as available treatment options for VL are limited.18–20 Therefore, the risk factors associated with increasing rate of relapse need to be deciphered. In our study, we found that male gender, weight, and spleen size before the start of the treatment were the risk factors associated with relapse. Males were twice (OR = 2.336, 95% CI: 1.303–4.187) more likely to suffer from relapse than females.
We also found that patients with weight ≤ 30 kg at day 0 were two times (OR = 1.947, 95% CI: 1.163–3.262) more prone to suffer from relapse than those with weight > 30 kg. Our findings were similar to other studies where poor nutritional status was found to be a risk factor for relapse.16,17
Furthermore, larger spleen size has also been shown to be associated with increased chances of relapse.12,17,21 We found that patients with spleen size > 4 at the start of the treatment were two times (OR = 1.998, 95% CI: 1.204–3.316) more likely to suffer from relapse than subjects with spleen size ≤ 4. These predictive factors of relapse should be taken care of while following up these patients. Limitation of the study includes not using further molecular methods such as real-time qPCR in diagnosis and molecular monitoring of parasite kinetics with antileishmanial drugs to get the absolute parasite quantification, which would have more useful especially in predicting the relapse cases of VL.
Thus, single-dose AmBisome treatment has high efficacy with minimal AEs and should be continued in the elimination program. However, longer follow-up of these patients are needed as a significant number of patients relapsed between 6 and 12 months.
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