Efficacy and safety of levamisole in childhood nephrotic syndrome: A meta-analysis

Abstract

Present evidence regarding the efficacy and safety of levamisole in childhood nephrotic syndrome (NS), particularly the steroid-sensitive NS (SSNS), is limited. We searched relevant databases such as PubMed/MEDLINE, Embase, Google Scholar, and Cochrane CENTRAL till June 30, 2020. We included 12 studies for evidence synthesis (5 were clinical trials that included 326 children). The proportion of children without relapses at 6–12 months was higher in the levamisole group as compared to steroids (relative risk [RR]: 5.9 [95% Confidence interval (CI): 0.13–264.8], I² = 85%). Levamisole as compared to the control increased the proportion of children without relapses at 6–12 months (RR: 3.55 [95% CI: 2.19–5.75], I² = 0%). The GRADE evidence was of “very-low certainty” except for the comparison of levamisole with control, the latter being of “moderate certainty.” To conclude, levamisole given to children with SSNS is beneficial in preventing relapses and achieving remission as compared to placebo or low-dose steroids. Good-quality trials are needed to provide a robust evidence in this regard. PROSPERO Registration number: CRD42018086247.

Introduction

Childhood nephrotic syndrome (NS) is a common disease, usually treated with oral steroids.[1,2] Complete remission of proteinuria is observed in approximately 80% of children, and this type is also known as steroid-sensitive NS (SSNS). The remaining 20% fail to respond to initial steroid therapy. In the SSNS group, approximately 40%–50% of patients either develop frequent relapse NS (FRNS) or become steroid-dependent NS (SDNS).[1,3] These children may need repeat (or continuous) treatment with corticosteroids, making them vulnerable to glucocorticoid-associated adverse events. Several steroid-sparing agents such as cyclophosphamide, mycophenolate mofetil (MMF), levamisole, calcineurin inhibitors, or rituximab have been tried to prevent relapses and decrease steroid toxicity in these groups of children.[1,4,5,6] Among these drugs, levamisole is considered to be cost-effective and relatively safe, compared with other glucocorticoid-sparing agents. Previous randomized controlled trials (RCTs),[7,8,9] and retrospective studies,[10,11,12] have shown that levamisole may be beneficial in children having FRNS or SDNS. However, the present evidence for the use of levamisole in childhood NS is limited. Moreover, a recent multicentric RCT has shown that long-term use of levamisole in FRNS or SDNS prolongs the time to first relapse and prevents relapses of proteinuria during 1-year period.[13] We tried to synthesize the current evidence for the use of levamisole in childhood SSNS.

Objectives

The objectives of this study were to compare the efficacy and safety of levamisole versus other drugs/immunomodulators used in the treatment of children with SSNS.

Methods

Types of studies

We included controlled clinical trials (randomized and quasi-randomized) for evidence synthesis.

Study populations

Children from 1 to 18 years of age and having SSNS were included. Those with steroid-resistant NS, impaired renal function, steroid toxicity, intake of immunosuppressive agents other than levamisole, and serious infections (peritonitis, pneumonia, and cellulitis) were excluded.

Types of intervention

The intervention consisted of giving levamisole after achieving remission through daily steroid (prednisolone) therapy.

Types of comparison/control

The studies comparing levamisole with placebo or standard alternate-day prednisolone therapy or another immunosuppressive therapy were considered.

Outcomes

Primary

1. Proportions of children being relapse free at 6, 12, and 24 months

2. Time (months) taken to first relapse.

Secondary

1. Mean number of relapses per year

2. Side effects

3. Difference in the cumulative steroid dose between the two groups.

Outcome definitions

• Remission: Absence or trace proteinuria daily for 3 consecutive days.

• Relapse: Proteinuria ≥3+ daily for 3 consecutive days.

Database search

We searched relevant databases such as PubMed/MEDLINE, Embase, Google Scholar, Cochrane CENTRAL, and Clinical Trials Registry (Clinicaltrials.gov, and CTRI) till June 30, 2020. We used the following keywords: ((((((levamisole) OR non-corticosteroid immunosuppressive agents) OR steroid sparing agents)) AND ((((nephrotic syndrome) OR (steroid sensitive nephrotic syndrome) OR (frequently relapsing nephrotic syndrome) OR (steroid dependent nephrotic syndrome)) AND ((((children) OR infants) OR childhood) OR Pediatrics)). No language restrictions were applied. Two authors (GCB and NRM) evaluated the search strategy for identification of relevant studies.

Extraction of data

Two review authors (GC and BP) extracted the data with the help of a predesigned pro forma. Data extraction was done by two authors. The data which were extracted included name of the authors, year of publication, study setting, population type, dose of levamisole, and its duration. We also looked for the sources of funding for the study. We resolved any disagreement by discussion with another author (RRD).

Assessment of study quality or risk of bias

Study quality or risk of bias in the included studies was independently assessed by two review authors (RRD and GCB) by using the Cochrane Collaboration tool.[14] We resolved any disagreement by discussion with another author (NRM).

Data synthesis

For continuous data (time to remission and cumulative steroid dosage), the mean difference with 95% confidence interval (CI) was calculated. For dichotomous data (number of relapses and side events), relative risk (RR) with 95% CI was calculated. P <0.05 was taken as statistically significant. I² statistics was used to assess study heterogeneity. If heterogeneity was high (I² > 50%) or statistically significant, we tried to find out the cause. Review Manager (version 5.3) was the statistical software used.[15]

Publication bias

This was assessed by methods (construction of inverted funnel plot) described previously.[16] In case of publication bias, trim and fill method was used to estimate for missing or unreported studies and recalculate effect estimate.[17]

Certainty of evidence

This was assessed by construction of a table through the GRADEprofiler software (v. 3.2).[18,19] The certainty of evidence can be either high or moderate or low or very low.

Results

A total of 202 articles were obtained by applying the above-mentioned search strategy. After screening, 182 articles got excluded, and after full-text review, another 8 articles got excluded (ongoing study = 1, review articles or non-RCTs = 6, and evaluated interleukin levels after giving levamisole = 1), and finally, 12 articles were included in the evidence synthesis [Figure 1]. Six studies compared alternate-day levamisole with placebo or no treatment,[7,8,9,13,20,21,22] one compared alternate-day prednisolone (<0.5 mg/kg for 1 year) with alternate-day levamisole,[23] one compared levamisole with MMF,[24] one compared cyclophosphamide with levamisole,[25] and the other compared levamisole with cyclophosphamide and controls.[21] One study compared daily with alternate-day levamisole,[26] and the other compared daily levamisole with MMF.[27] Only one study had “low risk” of bias in all the domains, and the others had “unclear risk” of bias in one or more domains except for blinding (high risk of bias) [Figure 2]. The studies were conducted in the following countries: India (n = 4),[8,21,24,27] Sri Lanka (n = 2),[20,26] Saudi Arabia (n = 1),[23] Bangladesh (n = 1),[9] and Egypt (n = 1).[25] Three studies were multicentric in the USA, The Netherlands, and the UK.[7,13,22] The details of these studies are provided in Table 1.

Figure 1.

PRISMA flow diagram. PRISMA = Preferred Reporting Items for Systematic Reviews and Meta-Analyses

Figure 2.

Summary of the risk of bias assessment

Table 1.

Description of the included studies

Study ID	Study setting, Country	Population details	Intervention studied	Outcome measures	Additional details
Kudagammana et al., 2017[20]	Single center, Sri Lanka	n=76 (alternate-day levamisole group=42; control group=34) Age (years): 3-14 Inclusion criteria: Those treated with maintenance low dose (0.1-0.6 mg/kg) alternate-day oral prednisolone and alternate-day levamisole (2.5 mg/kg) for 2 years, and were relapse free during the preceding 6 months	Levamisole: Alternate day (2.5 mg/kg) for 1 year Control group: No treatment	Relapse rate and potential side effects	Open label randomized controlled trial. Allocation concealment and blinding not done. Small sample size
Donia et al., 2005[25]	Single center, Egypt	n=40 (levamisole=20, cyclophosphamide=20) Age (years)=3-15 Inclusion criteria: children with SDNS minimal change (biopsy proven)	Levamisole: 2.5 mg/kg on alternate days for 6 months Cyclophosphamide: IV 500 mg/m2/month for 6 months	Outcome indicators: Remission rate, duration of remission, side effects, and compliance with therapy	Randomization method not clear, blinding was not done. Small sample size
Abeyagunawardena et al., 2017[26]	Single center pilot study, Sri Lanka	n=58 Age (years): 4.5-14.2 (median=7.95) Inclusion criterion: SDNS cases treated with levamisole and low-dose alternate-day prednisolone (0.1-0.6 mg/kg) and relapsing >2 in the preceding 12 months to the date of enrollment	Patients received levamisole (2.5 mg/kg) daily with alternate-day prednisolone for 1 year	Difference in number of relapses, change in laboratory tests prior to and after daily levamisole treatment initiation	Cross over (same patient first received alternate day and later received daily levamisole) study. Small sample size
Sural et al., 2001[21]	Single center, India	n=85 Age (years)=2-16 Inclusion criteria: FRNS who are steroid sensitive	Levamisole: 2.5 mg/kg on alternate days for 6 months CPA group: CPA 2 mg/kg/day for 12 weeks Control group: No treatment	Relapse rate at 6 months and 12 months, adverse effects	Small sample, only conference abstract available
British association for paediatric nephrology (BAPN), 1991[7]	Multicenter, UK	n=61 (Levamisole=31, Control=30) Age (years): Levamisole group (mean age 8.3), placebo group (mean age 8.8) Inclusion criteria: FRNS who were fully steroid responsive and had steroid	Levamisole: 2.5 mg/kg on alternate days Placebo group: Placebo therapy in the same dose	Primary outcome: Efficacy (was the time to first relapse)	Randomization method, blinding not clear. Exclusion criteria not defined. Small sample size
Al-Saran et al., 2006[23]	Single center, Saudi Arabia	n=56 (levamisole group=32; control group=24) Age (years), (median, range): Levamisole group (8.4, 3.5-14.6), control group (7.2, 2.8-13.8) Inclusion criteria: SSNS and FRNS or SDNS who were in remission	Levamisole: 2–2.5 mg/kg on alternate days for 1 year Control group: Low dose Prednisolone on an alternate-day basis (<0.5 mg/kg) for 1 year after achieving remission	Total relapse rate and the cumulative dose of steroids for 1 year before and 1 year after levamisole therapy, therapy failure, side effects and cost of levamisole therapy	Single center, crossover study. Small sample size
Sinha et al., 2019[24]	Single center, India	n=149 (levamisole=73, MMF=76) Age (years): 6-18 Inclusion criteria: All frequently relapsing or steroid-dependent nephrotic syndrome	MMF: 750–1000 mg/m2 daily Levamisole: 2–2.5 mg/kg on alternate days for 1 year Prednisolone was discontinued by 2–3 months in both groups	Primary outcome: comparison of the number of relapses during 12 months of follow-up Secondary outcomes sustained remission, and treatment failure, cumulative Steroid received, and adverse events	Randomization by computer-generated random number sequence. Allocation concealment was done by sealed envelopes. Blinding was not done
Gruppen et al., 2018[13]	Multicenter: 13 sites in 6 countries (The Netherlands, Belgium, France, Italy, Poland, and India)	n=99 (levamisole=50, placebo=49) Age (years): 2-18 Inclusion criteria: FRNS children with or without steroid dependence	Levamisole: 2.5 mg/kg on alternate days with a maximum dose of 150 mg Placebo: Same dose and day schedule with placebo as Levamisole Prednisone as per Protocol	Time to relapse (primary endpoint), Occurrence of a prednisone-needing relapses (secondary endpoint), and safety	Individual unblinding took place when a relapse occurred (to give patients in the placebo group the possibility of receiving Levamisole) or at the end of the 12 months trial. Small sample size
Dayal et al., 1994[8]	Single center, India	n=61 (levamisole=33, control=28) Age (month): Levamisole (59.9±33.0), control (58.2±35.6) Inclusion criteria: SSNS with MCNS at the time of initial diagnosis or during a subsequent relapse	Levamisole 2–3 mg/kg/day twice a week for 1 year Control group: No treatment was given	Primary outcome: Duration of remission	Blinded for laboratory personnel but not patients or investigator. Both initial episode and relapse cases were included. Small sample size
Rashid et al., 1996[9]	Single center, Bangladesh	n=40 Age (year)=1-15 Inclusion criteria : FRNS who are steroid sensitive, SDNS	Levamisole: 2.5 mg/kg on alternate days for 6 months control group: Placebo on alternate days	Relapse rate at 6 months and 12 months, adverse effects	Small sample size
Weiss 1993[22]	Multicenter, USA	n=49 Age (years): 3-12 Inclusion criteria: FRNS who are steroid sensitive, SDNS	Levamisole group: levamisole 2.5 mg/kg twice weekly for 6 months Control group: placebo twice weekly	Relapse rate at 6 months and 12 months, adverse effects	Multicenter, prospective, randomized, open-label trial. Relapse rate on twice weekly levamisole was compared to placebo therapy. Small sample size, only conference abstract available
Singh et al., 2020[27]	Single center, India	n=42 Age (years): 2–14 Inclusion criterion: SDNS/FRNS	Levamisole group: levamisole 2.5 mg/kg daily for 11 year MMF group: Enteric coated MMF @ 1000 mg/m2 for 1 year	Primary outcomes: Percentage reduction in relapse frequency at 1 year Secondary outcomes: Adverse effects, change in relapse frequency proportion of patients who were relapse	Open label trial. Free, cumulative steroid dose and failure of trial medication

SDNS=Steroid-dependent nephrotic syndrome, FRNS=Frequent relapse nephrotic syndrome, MCNS=Minimal change nephrotic syndrome, SSNS=Steroid-sensitive nephrotic syndrome, MMF=Mycophenolate mofetil

Primary outcomes

Proportion of children being relapse free at 6, 12, and 24 months

Proportion of children without relapses at 6–12 months:

1. Levamisole versus steroids: Data from two studies were pooled.[9,23] Levamisole group had a greater number of patients with sustained remission/without relapse (RR: 5.9 [95% CI, 0.13–264.8], I² = 85%) [Figure 3]

2. Levamisole versus placebo or no treatment: Data from three studies were pooled.[7,13,21] Levamisole group had a greater number of patients with sustained remission/no relapses (RR: 3.55 [95% CI, 2.19–5.75], I² = 0%) [Figure 3]

3. Levamisole versus steroids/placebo or no treatment: Data from three studies were pooled.[7,13,21] Levamisole group had a greater number of patients with sustained remission/no relapses (RR: 3.25 [95% CI, 1.86–5.67], I² = 31%) [Figure 3]. One study stratified the groups based on steroid dependence/frequent relapsers;[13] however, the difference was not statistically significant (RR: 0.56 [95% CI, 0.21–1.47])

4. Levamisole versus cyclophosphamide: Two studies reported this outcome.[21,25] No significant difference in the proportion of patients in sustained remission was found (RR: 1.11 [95% CI, 0.58–2.14]) even 6 months after stopping medications. The authors used IV cyclophosphamide pulse therapy

5. Levamisole versus MMF: Two studies reported this outcome.[24,27] One study compared alternate-day levamisole with daily MMF (750 mg to 1000 mg/m²), but no significant intergroup difference was found (RR: 1.32 [95% CI, 0.68–2.57]).[24] Another study comparing daily levamisole with MMF[27] also found no intergroup difference in the number of relapses (RR: 0.78 [95% CI, 0.20–3.11]) over 1-year study follow-up.

Figure 3.

Proportions of SDND/FRNS children without relapses at 6–12 months (levamisole vs. steroids/placebo or no treatment). SDNS = Steroid-dependent nephrotic syndrome, FRNS = Frequent relapse nephrotic syndrome

Proportion of children without relapses at 24 months:

1. Levamisole versus steroids or placebo or no treatment: No study reported the proportion of patients without relapses at 24 months. In one study, the proportion of children without relapse at 30-month follow-up was not significantly different (RR: 2.07 [95% CI, 0.84–5.08]).[8] In this study, levamisole was given for a period of 12 months

2. Levamisole versus cyclophosphamide: One study found no significant difference (RR: 3 [95% CI, 0.34–26.45]).[26]

Time to first relapse

One study (n = 103) reported this outcome.[13] The Kaplan–Meier analysis showed no difference in relapse-free survival over the initial 100 days. However, after 100 days, the relapse-free survival was favoring levamisole (P = 0.015).

Secondary outcomes

Proportion of the children with frequent relapse nephrotic syndrome

None of the studies reported this outcome. One study reported failure of the therapy in 9.4% versus 50% in children who received levamisole and low-dose prednisolone, respectively.[23]

Cumulative steroid dose (mg)

One study reported a mean (standard deviation) cumulative prednisolone dose of 255 (157) mg versus 327 (96) mg (P < 0.0001) in children who received levamisole and low-dose steroids, respectively.[24] In another study, less number of patients required steroid in the levamisole group (RR: 0.77 [95% CI, 0.61–0.97]).

Side effects or adverse events

Gastrointestinal symptoms

Mild symptoms were reported in two studies.[7,23] However, no significant statistical difference was achieved between the patients receiving levamisole as compared to placebo or low-dose steroids (RR: 2.57 [95% CI, 0.28–24.01]) [Figure 4]. Studies comparing levamisole with MMF have shown comparable episodes of acute gastroenteritis [Supplementary Figure 1 ^{(661.4KB, tif)} ].

Figure 4.

Adverse events in SDNS/FRNS children receiving levamisole versus steroids/placebo or no treatment, SDNS = Steroid-dependent nephrotic syndrome, FRNS = Frequent relapse nephrotic syndrome

Leukopenia

This outcome was reported by all the studies comparing levamisole versus steroid/placebo or no treatment.[7,8,9,13,20,21] In five studies, leukopenia was not seen. One study found no significant intergroup difference (RR: 5.00 [95% CI, 0.61–41.28]) [Figure 4].[13] Pooled data from two studies found no significant difference (RR: 0.25 [95% CI, 0.04–1.48]).[21,25] Two studies compared levamisole with MMF, and the proportion of the patients developing leukopenia was comparable in two groups (RR: 0.64 [95% CI, 0.08–5.04], I² = 0%) [Supplementary Figure 1 ^{(661.4KB, tif)} ].

Infections

One study compared levamisole versus cyclophosphamide, and found no significant difference (RR: 1.17; 95% CI: 0.74–1.85) [Figure 5].[25]

Figure 5.

Adverse events in SDNS children receiving levamisole versus cyclophosphamide. SDNS = Steroid-dependent nephrotic syndrome

Reduced estimated glomerular filtration rate and antineutrophil cytoplasmic antibody- positive arthritis

One study found no significant intergroup difference (one patient in each group developed these adverse events) [Figure 4].[13]

Alopecia

One study compared levamisole versus cyclophosphamide,[25] and found no significant intergroup difference (RR: 0.14 [95% CI, 0.10–2.60]) [Figure 5].

Hepatotoxicity

One study compared levamisole versus cyclophosphamide,[25] and found no significant intergroup difference (RR: 0.33 [95% CI, 0.01–7.72]) [Figure 5].

Daily versus alternate-day Levamisole

A crossover study (n = 64) reported the impact of daily dose of levamisole compared to standard alternate-day dose over 12 months.[26] The mean number of relapse episodes was 2.8 in patients on alternate-day levamisole, and 1.3 in those on daily levamisole. The major drawback of the study was the crossover nature, and the outcomes studied were drug efficacy only (the adverse effects were not reported).

Publication bias

As the number of studies was <10 for the primary outcome, we could not construct an inverted funnel plot to assess publication bias.

Sensitivity analysis

We did a sensitivity analysis by analyzing studies with a high and low risk of bias for blinding, but there was no significant difference in the results [Supplementary Figures 2 ^{(433.4KB, tif)} and 3 ^{(377KB, tif)} ].

Certainty of evidence

It was of “very low certainty” (except of “moderate certainty” for comparison of levamisole with placebo or no treatment) [Table 2]. This was due to significant study limitations and significant heterogeneity among the studies.

Table 2.

Levamisole compared to placebo or standard care for nephrotic syndrome

Patient or population: Patients with nephrotic syndrome Settings: Hospital Intervention: Levamisole Comparison: Placebo or standard care
Outcomes	Illustrative comparative risks* (95% CI)		Relative effect (95% CI)	Number of participants (studies)	Certainty of the evidence (GRADE)
	Assumed risk placebo or standard care	Corresponding risk levamisole
Children without relapses: All	Study population		RR 3.25 (1.86-5.67)	326 (5 studies)	⊕⊕⊖⊖Lowa, b
	126 per 1000	409 per 1000 (234-713)
	133 per 1000	432 per 1000 (247-754)
Children without relapses: Levamisole versus steroids	Study population		RR 5.9 (0.13-264.28)	90 (2 studies)	⊕⊖⊖⊖Very lowb, c
	105 per 1000	621 per 1000 (14-1000)
	143 per 1000	844 per 1000 (19-1000)
Children without relapses: Levamisole versus placebo or no treatment	Study population		RR 3.55 (2.19-5.75)	236 (3 studies)	⊕⊕⊕⊖Moderateb
	133 per 1000	471 per 1000 (291-763)
	133 per 1000	472 per 1000 (291-765)

^aHigh or unclear risk of bias in included studies, ^bSmall sample size, ^cHeterogeneity in included studies. CI=Confidence interval, The justifications for the assumed risk (for e.g. median control group risk across studies) is given in footnotes. The corresponding risk (with its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI)

Discussion

Summary of findings

A total of 12 studies were included in the evidence synthesis. Our result indicated that levamisole given to children with SSNS led to a higher rate of sustained remission/without relapse. The certainty of evidence generated for primary outcome was of “very low” to “moderate” certainty.

The present systematic review shows that levamisole probably decreases the relapses in children with SDNS/FRNS course. Two studies compared levamisole with low-dose steroid,[9,23] while another three compared levamisole with placebo or no treatment.[7,13,20] In the study by the British Association of Pediatric Nephrology (BAPN),[7] the efficacy of levamisole became apparent after 40 days compared to another multicenter study,[13] where the beneficial effect was observed in 100 days. The possible reasons could be a longer steroid treatment in the later study as compared to that by BAPN. Moreover, children in the BAPN study also received cyclophosphamide before starting levamisole, which has been suggested to be more efficacious in children who had previously received an alkylating agent.[28] The authors in the multicenter study[13] also showed an inferior response of SDNS patients (mostly from Europe) as compared to FRNS patients (mostly from India) in the subgroup analysis. A possible genetic and immunological switch because of steroid dependency and preceding long-term steroid therapy were hypothesized by the authors.[13,29]

Levamisole stimulates T-helper cells (Th1 and Th2) by increasing expressions of interleukin-8 activity and thereof has immunomodulatory activity,[30] and it is considered safe and less expensive among available steroid-sparing agents for preventing relapses in SSNS.[31] Limited studies comparing levamisole with other immunosuppressive agents such as cyclophosphamide or MMF have shown comparable efficacy profile.[21,24,25,27] In one study comparing alternate-day levamisole with MMF, levamisole was found to be comparable with MMF, though the dose of MMF used was lower (750–1000 mg/m²). The same study in post hoc analysis reported longer remission with MMF compared to levamisole.

Retrospective studies and a single crossover study[26] have shown a beneficial role of higher dose of levamisole (daily) as compared to alternate-day dosage in FRNS/SDNS. The rational for using daily levamisole in these studies was shorter half-life of levamisole (5.2 h).[32] In another study, the authors concluded that daily levamisole can be considered when alternate-day therapy is unsatisfactory, without increase in any adverse events.[32] However, more evidence is needed in this regard.

The adverse effects of levamisole are mostly transient and mild, and usually disappear after discontinuation of the drug. The pooled analysis found no significant difference in the rate of adverse events as compared to long-term steroid therapy or control.

Limitations

The included studies were heterogeneous (patient selection, duration of drug therapy, outcome definitions, and drug dosage). Moreover, the number of studies comparing levamisole with cyclophosphamide or MMF is limiting.

Conclusion and Future Areas for Research

Levamisole increases the proportion of children with remission compared to steroids (very low certainty evidence) and placebo or no treatment (moderate certainty evidence) in children. Future, multicenter studies looking at the efficacy of levamisole with other second-line agents and addressing the above-mentioned limitation are required.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Supplementary Figure 1

Adverse events in FRNS/SDNS children receiving levamisole versus MMF. SDNS = Steroid-dependent nephrotic syndrome, FRNS = Frequent relapse nephrotic syndrome, MMF = Mycophenolate mofetil

Supplementary Figure 2

Subgroup analysis based on studies with high risk of bias and low risk of bias for blinding (levamisole vs. steroids/placebo or no treatment)

Supplementary Figure 3

Subgroup analysis based on single-center versus multicentric studies (levamisole vs. steroids/placebo or no treatment)