BackgroundPlasmodium falciparum encoded histidine rich protein (HRP2) based malaria rapid diagnostic tests (RDTs) are used in India. Deletion of pfhrp2 and pfhrp3 genes contributes to false negative test results, and large numbers of such deletions have been reported from South America, highlighting the importance of surveillance to detect such deletions.MethodsThis is the first prospective field study carried out at 16 sites located in eight endemic states of India to assess the performance of PfHRP2 based RDT kits used in the national malaria control programme. In this study, microscopically confirmed P. falciparum but RDT negative samples were assessed for presence of pfhrp2, pfhrp3, and their flanking genes using PCR.ResultsAmong 1521 microscopically positive P. falciparum samples screened, 50 were negative by HRP2 based RDT test. Molecular testing was carried out using these 50 RDT negative samples by assuming that 1471 RDT positive samples carried pfhrp2 gene. It was found that 2.4% (36/1521) and 1.8% (27/1521) of samples were negative for pfhrp2 and pfhrp3 genes, respectively. However, the frequency of pfhrp2 deletions varied between the sites ranging from 0–25% (2.4, 95% CI; 1.6–3.3). The frequency of both pfhrp2 and pfhrp3 gene deletion varied from 0–8% (1.6, 95% CI; 1.0–2.4).ConclusionThis study provides evidence for low level presence of pfhrp2 and pfhrp3 deleted P. falciparum parasites in different endemic regions of India, and periodic surveillance is warranted for reliable use of PfHRP2 based RDTs.

SummaryTwo different Toll-like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR-2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR-9 has been shown to promote a host-protective response. However, whether there is a relationship between the interaction of LPG and TLR-2, on one hand, with the effect of TLR-9, on the other hand, remains unknown. In this study, we report that in-vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR-9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti-LPG as well as anti-TLR-2 antibodies prevents this reduction of TLR-9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR-9 expression, which is shown to be mediated by transforming growth factor (

TLRs recognize pathogen-expressed Ags and elicit host-protective immune response. Although TLR2 forms heterodimers with TLR1 or TLR6, recognizing different ligands, differences in the functions of these heterodimers remain unknown. In this study, we report that in Leishmania major-infected macrophages, the expression of TLR1 and TLR2, but not TLR6, increased; TLR2–TLR2 association increased, but TLR2–TLR6 association diminished. Lentivirus-expressed TLR1–short hairpin RNA (shRNA) or TLR2–shRNA administration reduced, but TLR6–shRNA increased L. major infection in BALB/c mice. Corroboratively, Pam3CSK4 (TLR1–TLR2 ligand) and peptidoglycan (TLR2 ligand) increased L. major infection but reduced TLR9 expression, whereas pegylated bisacycloxypropylcysteine (BPPcysMPEG; TLR2–TLR6 ligand) reduced L. major number in L. major-infected macrophages, accompanied by increased TLR9 expression, higher IL-12 production, and inducible NO synthase expression. Whereas MyD88, Toll/IL-1R adaptor protein, and TNFR-α–associated factor 6 recruitments to TLR2 were not different in Pam3CSK4-, peptidoglycan-, or BPPcysMPEG-treated macrophages, only BPPcysMPEG enhanced p38MAPK and activating transcription factor 2 activation. BPPcysMPEG conferred antileishmanial functions to L. major-infected BALB/c-derived T cells in a macrophage–T cell coculture and in BALB/c mice; the protection was TLR6 dependent and IL-12 dependent, and it was accompanied by reduced regulatory T cell number. BPPcysMPEG administration during the priming with fixed L. major protected BALB/c mice against challenge L. major infection; the protection was accompanied by low IL-4 and IL-10, but high IFN-γ productions and reduced regulatory T cells. Thus, BPPcysMPEG, a novel diacylated lipopeptide ligand for TLR2–TLR6 heterodimer, induces IL-12–dependent, inducible NO synthase–dependent, T-reg–sensitive antileishmanial protection. The data reveal a novel dimerization partner-dependent duality in TLR2 function.

The aim of this study was to evaluate the antiviral potential of methanolic extract (ME) of Achyranthes aspera, an Indian folk medicine and one of its pure compound oleanolic acid (OA) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The ME possessed weak anti-herpes virus activity (EC50 64.4μg/ml for HSV-1 and 72.8μg/ml for HSV-2). While OA exhibited potent antiherpesvirus activity against both HSV-1 (EC50 6.8μg/ml) and HSV-2 (EC50 7.8μg/ml). The time response study revealed that the antiviral activity of ME and OA is highest at 2-6h post infection. The infected and drug-treated peritoneal macrophage at specific time showed increased level of pro-inflammatory cytokines (IL6 and IL12). Further, the PCR of DNA from infected cultures treated with ME and OA, at various time intervals, failed to show amplification at 48-72h, similar to that of HSV infected cells treated with acyclovir, indicating that the ME and OA probably inhibit the early stage of multiplication (post infection of 2-6h). Thus, our study demonstrated that ME and OA have good anti-HSV activity, with SI values of 12, suggesting the potential use of this plant.

Commercial malaria rapid diagnostic tests (RDTs) detect P. falciparum histidine rich protein 2 (PfHRP2) and cross react with PfHRP3, a structural homologue. Here, we analysed natural variations in PfHRP2 and PfHRP3 sequences from Indian isolates and correlated these variations with RDT reactivity. A total 1392 P. falciparum positive samples collected from eight endemic states were PCR amplified for Pfhrp2 and Pfhrp3 genes and were sequenced. The deduced protein sequences were analysed for repeat variations and correlated with RDT reactivity. Out of 1392 PCR amplified samples, a single sample was Pfhrp2 negative and two samples were Pfhrp3 negative. Complete Pfhrp2 and Pfhrp3 sequences were obtained for 769 samples and 750 samples, respectively. A total of 16 distinct repeat motifs were observed for Pfhrp2 and 11 for Pfhrp3, including some new repeat types. No correlation was found between variations in the size of Pfhrp2 repeat types 2 and 7, nor between any combinations of repeat motifs, and performance of a commercial RDT at low parasite densities. The findings suggest that sequence diversity in Pfhrp2 and Pfhrp3 genes in Indian isolates is not likely to negatively influence performance of currently used PfHRP2 RDTs.

SummaryToll-like receptors (TLRs) recognize pathogen-associated molecular patterns and results in innate immune system activation that results in elicitation of the adaptive immune response. One crucial modulator of the adaptive immune response is CD40. However, whether these molecules influence each other's expression and functions is not known. Therefore, we examined the effects of TLRs on CD40 expression on macrophages, the host cell for the protozoan parasite Leishmania major. While polyinosinic-polycytidylic acid [poly (I:C)], a TLR-3 ligand, lipopolysaccharide (LPS), a TLR-4 ligand, imiquimod, a TLR-7/8 ligand and cytosine-phosphate-guanosine (CpG), a TLR-9 ligand, were shown to enhance CD40 expression, CD40 stimulation enhanced only TLR-9 expression. Therefore, we tested the synergism between CD40 and CpG in anti-leishmanial immune response. In Leishmaniainfected macrophages, CpG was found to reduce CD40-induced extracellular stress-regulated kinase (ERK)1/2 activation; with the exception of interleukin (IL)-10, these ligands had differential effects on CD40-induced IL-1α, IL-6 and IL-12 production. CpG significantly enhanced the anti-leishmanial function of CD40 with differential effects on IL-4, IL-10 and interferon (IFN)-γ production in susceptible BALB/c mice. Thus, we report the first systematic study on CD40-TLR cross-talk that regulated the experimental L. major infection.

Salmonella enterica serovar Typhimurium has been extensively exploited as live attenuated vaccines (LAV) which generally confers better protection than killed or subunit vaccines. However, many LAV are limited by their inherent ability to access systemic organs in many of the vaccinated hosts, especially those which are immunocompromised. We evaluated the efficacy of a live-attenuated SPI2-deficient (ΔssaV) S. Typhimurium vaccine candidate (MT13) that additionally devoids the ferric uptake regulator (fur). We used specific pathogen free (SPF) streptomycin-pretreated mouse colitis model that included healthy C57BL/6 and immunocompromised iNos −/−, IL10−/− and CD40L−/− in the background of C57BL/6 mice to assess the efficacy of developed vaccine candidate. In our study, the S. Typhimurium MT13 strain was established as a safe vaccine candidate to be administered in immunocompromised mice as it was found to be systemically attenuated without conferring significant pathological signs and growth defect within the host. In bacterial challenge experiment, the MT13-vaccinated C57BL/6 mice were protected from subsequent wild-type S. Typhimurium infection by inducing proficient mucosal immunity. The MT13 strain elicited efficient O-antigen specific mucosal secretory IgA associated protective response which was comparable with its parental ssaV mutant. Vaccination with MT13 also showed proficient T-cell activation in host mice; which has direct relation with pathogen clearance from host tissues. Collectively, these data implicate the possible application of SPI-2 deficient fur mutant (MT13) as a novel live attenuated vaccine strain with adept immunogenicity and improved safety, even in immunocompromised hosts. Further, this vaccine candidate can be employed to express heterologous antigens targeted against several other diseases, especially related to enterocolitic pathogens.

No abstract