A randomised controlled trial to compare the efficacy, safety, and tolerability of low dose, short course primaquine in adults with uncomplicated P. vivax malaria in two hospitals in India

Background: Plasmodium vivax remains a major challenge for malaria control and elimination due to its ability to cause relapsing illness. To prevent relapses the Indian National Center for Vector Borne Diseases Control (NCVBDC) recommends treatment with primaquine at a dose of 0.25 mg/kg/day provided over 14 days. Shorter treatment courses may improve adherence and treatment effectiveness. Methods: This is a hospital-based, randomised, controlled, open-label trial in two centres in India. Patients above the age of 16 years, with uncomplicated vivax malaria, G6PD activity of ≥ 30% of the adjusted male median (AMM) and haemoglobin levels ≥ 8 g/dL will be recruited into the study and randomised in a 1:1 ratio to receive standard schizonticidal treatment plus 7-day primaquine at 0.50 mg/kg/day or standard care with schizonticidal treatment plus 14-day primaquine at 0.25 mg/kg/day. Patients will be followed up for 6 months. The primary endpoint is the incidence risk of any P. vivax parasitaemia at 6 months. Safety outcomes include the incidence risk of severe anaemia (haemoglobin 25% fall in haemoglobin and an acute drop in haemoglobin of > 5 g/dL during primaquine treatment. Discussion: This study will evaluate the efficacy and safety of a 7-day primaquine regimen compared to the standard 14-day regimen in India. Results from this trial are likely to directly inform national treatment guidelines. Trial registration: Trial is registered on CTRI portal, Registration No: CTRI/2022/12/048283

Comparative analysis of cellular immune responses in treated Leishmania patients and hamsters against recombinant Th1 stimulatory proteins of Leishmania donovani

Our prior studies demonstrated that cellular response of T helper 1 (Th1) type was generated by a soluble antigenic fraction (ranging from 89.9–97.1 kDa) of Leishmania donovani promastigote, in treated Leishmania patients as well as hamsters and showed significant prophylactic potential against experimental visceral leishmaniasis (VL). Eighteen Th1 stimulatory proteins were identified through proteomic analysis of this subfraction, out of which fifteen were developed as recombinant proteins. In the present work, we have evaluated these fifteen recombinant proteins simultaneously for their comparative cellular responses in treated Leishmania patients and hamsters. Six proteins viz. elongation factor-2, enolase, aldolase, triose phosphate isomerase, protein disulfide isomerase and p45 emerged as most immunogenic as they produced a significant lymphoproliferative response, nitric oxide generation and Th1 cytokine response in PBMCs of treated patients and lymphocytes of treated hamsters. The results suggested that these proteins may be exploited for developing a successful poly-protein and/or poly-epitope vaccine against VL

Leishmania-specific IgG and its isotypes IgG1 and IgG2 responses.

<p>Serum samples were collected from different groups of hamsters at designated time points and assayed for rLdSir2RP—specific IgG, IgG1, and IgG2 levels by ELISA. Antibody responses in rLdSir2RP vaccinated hamsters in comparison to the unimmunized infected hamsters on days 45, 60 and 90 p.c. The IgG1 were observed to be elevated by 1 to 2 fold in infected control group in comparison to the group of immunized hamsters with rLdSir2RP at the time interval of Days 45(a), d60(b) and d90(c). There was significant elevation of IgG2 level (by 3.5 to 4 folds) in group of hamsters immunized with rLdSir2RP at the time interval of Days 45(a), d60(b)and d90(c). Data are presented as the absorbance at 492 nm and are means ± SE for 3–4 hamsters per group in triplicate wells. Significance values indicate the difference between the vaccinated group and infected group (***, p<0.001).</p

Parasite burden.

<p>Clinical outcomes following <i>L</i>. <i>donovani</i> challenge in hamsters immunized with rLdSir2RP+BCG. On day 21 after the booster, the hamsters of infected, BCG alone and vaccinated groups were challenged intracardially with 10^<b>7</b> metacyclic promastigotes of <i>L</i>. <i>donovani</i>. Following parameters observed Body weight (a), spleen weight (b), liver weight (c), Parasite burden (no. of amastigotes per 100 cell nuclei) in the spleen(d), liver(e), and bone marrow (f) on days 45 and 60 p.c. Significance values indicate the difference between the vaccinated groups and infected group (***, p<0.001). Data represent mean values standard errors (SE) at the designated time points.</p

Expression of recombinant LdSir2RP in E. coli Rosetta strain and its purification.

<p>Expression and purification of rLdSir2RP (1A, IB) in prokaryotic cells, Whole Cell Lysate (WCL) of transformed <i>E</i>. <i>coli</i> separated on 12% acrylamide gel and stained with Coomassie blue, M: Molecular wt. markers; Lane 1: WCL before IPTG induction; lane 2: WCL after IPTG (1.0 mM) induction at 37°C. Lane 5: eluted protein (1B). Western blot analysis using anti-rLdSir2RPAb in uninduced WCL, induced WCL and SLD—M: Mol wt marker, Lane 1: uninduced WCL, Lane 2: induced WCL (1C).</p

Th2 cytokine mRNA expression.

<p>Th2 cytokines mRNA expression profile analysis of normal and immunized hamsters on days 45 p.c.and 90 p.c were checked by quantitative real-time RT-PCR. The expression of Th2 cytokines viz. IL4 (a,b), IL10 (c,d), TGFß (e,f) was also observed to be ~3.00 folds higher (p<0.001) in the Infected hamsters as compare to vaccinated group as well as control groups. Significance values indicate the difference between the vaccinated group and infected group (***, p<0.001), vaccinated group and BCG group (***, p<0.001), vaccinated group and normal group (*, p<0.05).</p

Cellular responses of rLdSir2RP of L.donovani in vaccinated hamsters.

<p>Nitric oxide production (μM) (a, b, c) by J774A.1 cell line. The J774 A.1 cells were primed with the supernatants of stimulated lymphocytes (3 days with mitogen and 5 days with Ags) of normal, infected and vaccinated hamsters in response to rLdSir2RP, SLD and LPS respectively at 10 μg/ml each. The estimation of NO production was done using Greiss reagent in supernatants collected from macrophage cultures 24 h after incubation and the absorbance of the reaction product was measured at 540 nm. XTT response (d, e, f) of mononuclear cells (lymph nodes) from normal, <i>L</i>. <i>donovani</i> infected and vaccinated hamsters in response to Con A, SLD and rLdSir2RP at 10 μg/ml each Proliferation was represented as mean OD. The data represent the means of triplicate wells ± S.D. Significance values indicate the difference between the SLD and rLdSir2RP stimulation (**, p < 0.01; ***, p<0.001).</p