BackgroundLeishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD.MethodsA genomic DNA sample was applied to determine the assay analytical sensitivity. The cross-reactivity of the assay was tested by DNA of Leishmania spp. and of pathogens considered for differential diagnosis. The clinical performance of the assay was evaluated on LD positive and negative samples. All results were compared with real-time PCR. To allow the use of the assay at field settings, a mobile suitcase laboratory (56 × 45.5 × 26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh.ResultsThe LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature (42 °C) in 15 min. The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD-negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100 % agreement. The suitcase laboratory was successfully operated at the local hospital by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method (SpeedXtract), in which a simple fast lysis protocol was applied. Moreover, All reagents were cold-chain independent.ConclusionsThe mobile suitcase laboratory using RPA is ideal for rapid sensitive and specific detection of LD especially at low resource settings and could contribute to VL control and elimination programmes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1572-8) contains supplementary material, which is available to authorized users.

Highlights d Stunted children have distinct gut phage communities relative to non-stunted ones d In vitro, phages regulate bacteria isolated from children younger than 23 months d Non-stunted children harbor more temperate phages, irrespective of age d Bacteria in stunted children have more traits linked to human disease and metabolism

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.

Vaccine development for vector-borne pathogens may be accelerated through the use of relevant challenge models, as has been the case for malaria. Because of the demonstrated biological importance of vector-derived molecules in establishing natural infections, incorporating natural challenge models into vaccine development strategies may increase the accuracy of predicting efficacy under field conditions. Until recently, however, there was no natural challenge model available for the evaluation of vaccine candidates against visceral leishmaniasis. We previously demonstrated that a candidate vaccine against visceral leishmaniasis containing the antigen LEISH-F3 could provide protection in preclinical models and induce potent T-cell responses in human volunteers. In the present study, we describe a next generation candidate, LEISH-F3+, generated by adding a third antigen to the LEISH-F3 di-fusion protein. The rationale for adding a third component, derived from cysteine protease (CPB), was based on previously demonstrated protection achieved with this antigen, as well as on recognition by human T cells from individuals with latent infection. Prophylactic immunization with LEISH-F3+formulated with glucopyranosyl lipid A adjuvant in stable emulsion significantly reduced both Leishmania infantum and L. donovani burdens in needle challenge mouse models of infection. Importantly, the data obtained in these infection models were validated by the ability of LEISH-F3+/glucopyranosyl lipid A adjuvant in stable emulsion to induce significant protection in hamsters, a model of both infection and disease, following challenge by L. donovani–infected Lutzomyia longipalpis sand flies, a natural vector. This is an important demonstration of vaccine protection against visceral leishmaniasis using a natural challenge model.

BackgroundPost-kala-azar dermal leishmaniasis (PKDL) is a sequel to visceral leishmaniasis (VL), which is found in VL-endemic countries including Bangladesh. Because of these enigmatic cases, the success of the National Kala-azar Elimination Program is under threat. To date, diagnostic methods for PKDL cases in endemic regions have been limited to clinical examination and rK39 test or microscopy, and a suitable and accurate alternative method is needed. In this study, we investigated the application of real-time polymerase chain reaction (PCR) as a potential method for diagnosis of PKDL in comparison with microscopy.MethodsNinety-one suspected macular PKDL cases from Mymensingh district, Bangladesh, were enrolled in the study after diagnosis by clinical examination and an rK39 strip test. All of them responded after completion of the treatment with miltefosine. During enrollment, a skin biopsy was done for each patient, and both microscopy and real-time PCR were performed for detection and quantification of Leishmania donovan body (LDB) and LD DNA, respectively.ResultsReal-time PCR detected 83 cases among all suspected PKDL patients, with an encouraging sensitivity of 91.2% (83.4%–96.1%), whereas microscopy showed 50.6% (39.9%–61.2%) sensitivity. Among all suspected PKDL cases, 42 cases were positive in both microscopy and qPCR, whereas 41 cases were detected as positive through qPCR only.ConclusionsThis study provides evidence that real-time PCR is a promising tool for diagnosis of PKDL in endemic regions. In addition to diagnosis, the quantitative ability of this method could be further exploited for after-treatment prognosis and cure assessment of PKDL cases.

BackgroundRecombinant fusion proteins are now commonly used to detect circulating antibodies for the serodiagnosis of visceral leishmaniasis (VL) in Asia, Africa and the Americas. Although simple, these tests still require blood collection and their use in remote settings can be limited due to the need of collection devices, serum fractionation instrument and generation of biohazardous waste. The development of an accurate and non-invasive diagnostic algorithm for VL, such as could be achieved with urine, is desirable.MethodsWe enrolled 87 VL patients and 81 non-VL individuals, including 33 healthy endemic controls, 16 healthy non-endemic controls, 16 disease controls and 16 tuberculosis (TB) patients. We compared the efficacy of recombinant antigens rK28, rK39 and rKRP42 for the diagnosis of VL when either serum or urine were used to develop antibody-detection ELISA.ResultsAs expected, each of the antigens readily detected antibodies in the serum of VL patients. rK28 ELISA showed the highest sensitivity (98.9 %), followed by rK39 and rKRP42 ELISA (97.7 and 94.4 %, respectively); overall specificity was > 96 %. When urine was used as the test analyte, only a marginal drop in sensitivity was observed, with rK28 ELISA again demonstrating the greatest sensitivity (95.4 %), followed by rK39 and rKRP42 ELISA, respectively. Again, the overall specificity was > 96 %.ConclusionsOur data indicate the potential for using urine in the diagnosis of VL. Detection of antibodies against rK28 demonstrated the greatest sensitivity. Together, our results indicate that rK28-based antibody detection tests using urine could provide a completely non-invasive tool amenable for diagnosis of VL in remote locations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1667-2) contains supplementary material, which is available to authorized users.

Background Visceral leishmaniasis (VL) caused by dimorphic Leishmania species is a parasitic disease with high socioeconomic burden in endemic areas worldwide. Sustaining control of VL in terms of proper and prevailing immunity development is a global necessity amid unavailability of a prophylactic vaccine. Screening of experimental proteome of the human disease propagating form of Leishmania donovani (amastigote) can be more pragmatic for in silico mining of novel vaccine candidates. Methods By using an immunoinformatic approach, CD4+ and CD8+ T cell-specific epitopes from experimentally reported L. donovani proteins having secretory potential and increased abundance in amastigotes were screened. A chimera linked with a Toll-like receptor 4 (TLR4) peptide adjuvant was constructed and evaluated for physicochemical characteristics, binding interaction with TLR4 in simulated physiological condition and the trend of immune response following hypothetical immunization. Results Selected epitopes from physiologically important L. donovani proteins were found mostly conserved in L. infantum, covering theoretically more than 98% of the global population. The multi-epitope chimeric vaccine was predicted as stable, antigenic and non-allergenic. Structural analysis of vaccine-TLR4 receptor docked complex and its molecular dynamics simulation suggest sufficiently stable binding interface along with prospect of non-canonical receptor activation. Simulation dynamics of immune response following hypothetical immunization indicate active and memory B as well as CD4+ T cell generation potential, and likely chance of a more Th1 polarized response. Conclusions The methodological approach and results from this study could facilitate more informed screening and selection of candidate antigenic proteins for entry into vaccine production pipeline in future to control human VL.

Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus, E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus, ten bacterial cells for E. coli and two oocysts for C. parvum. The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels.