A Pilot Study for Capacity Building for a Multi-centre, Randomized Trial for Treatment of Kala Azar in Bangladesh
Condition: Visceral Leishmaniasis
MeSH terms: Leishmaniasis; Leishmaniasis, Visceral
Study type: Interventional

Summary: 
      Kala azar (KA) or visceral leishmaniasis (VL) is endemic in several districts of Bangladesh
      with the highest incidence in Mymensingh, Pabna and Tangail districts. ICDDR,B is involved in
      a project for improving the surveillance of KA in Trishal, Mymensingh since 2005. Improvement
      of case detection is necessary for both surveillance purposes and better control. The aims of
      this pilot study are to assess some newer techniques for diagnosis of KA using blood and
      urine samples of suspected cases; and evaluate response to treatment with sodium
      stibogluconate to which resistance has been reported in India, considered to be a part of the
      same zone harboring the disease agent Leishmania donovani and transmitted by the same vector
      Phlebotomas argentipes (sand-fly). No data is currently available on response to sodium
      stibogluconate in KA patients in Bangladesh. Although a number of new drugs have been
      evaluated in the treatment of KA in India and Kenya, no trial has so far been conducted in
      Bangladesh. A team of researchers from GlaxoSmithKline (UK) had recently visited Bangladesh
      to evaluate if it would be possible to conduct a Phase-III clinical trial with sitamaquine.
      They interacted with scientists of ICDDR,B and expressed their interest to help develop
      ICDDR,B's capacity in order to include Bangladesh as one of the sites for the planned,
      multi-centre, Phase-III trial of sitamaquine; India and Nepal are two other possible sites
      for the trial. The aims of the proposed study are to train physicians and laboratory
      personnel in preparation for the future drug trial(s) on KA as well as to compare different
      tests for its diagnosis that might improve case detection at the field level and used for
      research purposes. The investigators will also examine in greater detail the different
      Leishmania species circulating in the area of Mymensingh and whether treatment failure and
      occurrence of Post Kala azar Dermal Leishmaniasis (PKDL) is associated with certain species.
    

Detailed description: 
      Specific Aims:

      Primary objective: To develop capacity to evaluate newer regimens for treatment of kala azar
      in Bangladesh.

      Secondary objectives:

        1. To evaluate novel diagnostic tests for kala azar including rK39 dipstick test in serum;
           KATEX in urine; and PCR (polymerase chain reaction) in blood and clinical samples.

        2. To evaluate response to treatment with 28 injections of SAG using PCR in blood samples
           and clinical samples.

        3. To transfer the mini-exon PCR-RFLP technique to ICDDR,B and to characterize the most
           prevalent Leishmania species in the area of Mymensingh and their response to standard
           treatment with SAG.

        4. We will also analyze whether certain genotypes are associated with the occurrence of
           PKDL and whether the mini-exon PCR is a suitable marker for monitoring the intra and
           interspecies genetic evolution of Leishmania species.

      Research Design and Methods:

      The patients for the proposed study were selected from three upazilla health complexes in
      Mymensingh per discussion with the government agencies working in these areas. If necessary,
      we would go for active surveillance to enroll required number of subjects for the study.

      Patients

      In total, 200 consecutive cases (age >5 years) meeting the following diagnostic criteria for
      KA were enrolled into the study: (i) fever for >2 weeks; (ii) at least one of the the
      following criteria- splenomegaly, darkening of the skin, and weight loss; and (iii) a
      positive rK39 dipstick test. Exclusion criteria will include: (1) children under five years
      of age, (2) pregnant women, and (3) patients who are suffering simultaneously from any other
      serious illness which is unrelated to kala azar. Patients (or their parents/guardians in the
      event of minors) meeting the above criteria, was explained about the risk of treatment with
      SAG and the risk involved in splenic aspiration procedures by expert physicians. Informed
      consent (verbal or written) was then obtained from the patients (or their parents/guardians
      in the event of minors) before inclusion into the study. One hundred healthy individuals
      living in the same area were enrolled as controls for evaluating PCR and other diagnostic
      tests of KA. Our field workers visited houses in the community and collect blood samples from
      the healthy volunteers with their informed consent.

      Study procedures and assessments

      Splenic aspiration:

      Splenic aspiration was performed in 200 consecutive patients who had been presented with
      clinical features of KA with a positive rK39 dipstick test. Splenic aspiration was performed
      for confirmation of diagnosis on study day 1, and then again on day 29 to assess cure from
      the disease. Thus, when a subject was found to be positive by rK39 dipstick test, standard
      pre-procedure tests for splenic aspiration e,g. hemoglobin estimation, bleeding time,
      clotting time, and platelet counts, were performed. Splenic aspiration was performed only in
      those who were not severely anemic (Hb ≥ 6 mg/dl), and had platelet counts of >50,000/micro
      liter of blood. Prothrombin time was also be checked before splenic aspiration and those with
      a PT more than 4 seconds prolonged were not be eligible for the study. All necessary
      treatment was provided in the event of hemorrhage following the procedure, including blood
      transfusion. If necessary, the patients also be transported to Mymensingh Medical College
      Hospital, for more specialized treatment and support.

      Treatment and follow up

      In Bangladesh, intramuscular administration of sodium antimony gluconate (SAG; Albert David
      Ltd., India), in a dose of 20 mg/kg body weight, with a maximum dose of 850 mg/day, for
      twenty eight days is the current standard treatment for patients with KA. The pulse and
      respiratory rates, blood pressures, and temperature were monitored at 12 hourly intervals
      during this treatment period. EKG were performed if clinically indicated. The treatment was
      stopped if signs of severe toxicity (cardiotoxicity, renal failure, etc) develop, and the
      patient had been treated with standard dose of amphotericin B.

      Hospitalization: All patients under treatment was offered to stay at Community-based Medical
      College Hospital for the entire period of treatment with SAG. The patients had been
      hospitalized for treatment with amphotericin B in case of treatment failure or toxicity with
      SAG.

      Follow up: At the end of treatment, the patients was evaluated for cure by physical
      examination and splenic aspiration. The subjects had been following for six months as shown
      in Scheme-2.

      Diagnostic tests to be evaluated

      rK39 dipstick test A finger-prick blood specimen was collected in capillary tubes for rK39
      dipstick test. This method is rapid and time saving.

      Parasitological diagnosis

      Splenic aspirate was used for parasitological diagnosis. A slide of splenic aspirates was
      prepared for Giemsa staining. Two lab technicians from ICDDR,B was trained on staining
      techniques and reading of the slides. Randomly selected slides had been sent to reference
      labs elsewhere for Quality Control.

      Polymerase chain reaction (PCR)

      DNA of Leishmania donovani in the collected blood samples had been detected using the methods
      described by Salotra et al.

      Sample collection and DNA isolation: Collected blood specimen was transported to the
      Parasitology Laboratory of ICDDR,B, and transferred to 4°C and processed generally on the
      same day. DNA was extracted from 0.2 ml of blood using a QIAamp DNA blood mini kit (Qiagen).

      PCR amplification: DNA from cultured parasites (1 ng) and from clinical samples (100 ng) were
      taken for amplification using the LdI primers described above. The reaction mixture (50 µl)
      contained 10 mM Tris-HCl (pH 8.3) 50 mM KCl, 1.5 mM MgCl2, a 200 µM concentration of each
      deoxynucleoside triphosphate, 50 ng of each primer, and 1.25 U of Taq DNA polymerase (Gibco
      BRL). Each reaction mixture was overlaid with mineral oil, and amplification was performed in
      a thermal cycler (Perkin-Elmer, Warrington, Great Britain) programmed for 40 cycles of
      denaturation at 94°C for 1 min, annealing at 45°C for 1 min, and extension at 72°C for 2 min,
      preceded by an initial denaturation of 2 min at 94°C. Final extension was for 3 min at 72°C.
      Products was analyzed by electrophoresis in 1% agarose gel containing ethidium bromide (0.5
      µg/ml) in TAE buffer (0.04 M Tris acetate, 0.001 M EDTA) and photographed under UV
      illumination.

      Mini-exon PCR-RFLP: The mini-exon PCR-RFLP had been developed by the Swiss Tropical Institute
      (STI) (Marfurth et al. 2003).

      Sample Size Calculation and Outcome Variable(s):

      The sample size of 200 cases and 100 controls is based on practical considerations for
      meeting the primary objective of capacity development and the study is not formally powered
      for hypothesis testing. The emphasis for investigating sensitivity and specificity of the new
      diagnostic tests will be on presenting descriptive statistics and confidence intervals.

      Data Analysis:

      The sensitivity (proportion of correctly identified true positive results) and specificity
      (proportion of correctly identified true negative results) was calculated for each diagnostic
      test, and 95% confidence intervals for these proportions had been calculated using Wilsons
      method.

      The positive and negative predictive values was also be calculated using the method described
      by Altman et al. The positive predictive value is the proportion of patients with a positive
      result for the experimental diagnostic test who are correctly diagnosed by parasitological
      examination. Similarly the negative predictive value is the proportion of patients with a
      negative diagnostic test result who are correctly diagnosed by parasitological examination.
      The 95% CI for the positive and the negative predictive values had been determined. These
      parameters would allow the comparison of each diagnostic test with the 'gold standard'
      parasitological examination.
    

Intervention name(s): Randomized trial for treatment of kala azar in Bangladesh
Intervention type(s): Drug
Intervention description(s): dose of SAG 20 mg/kg body weight, with a maximum dose of 850 mg/day, for 28 days
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: kala azar; visceral leishmaniasis; diagnosis; drug resistance; Therapeutics; Capacity Building