Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients.

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL. METHODS: In this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood. CONCLUSION: These results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.

Design, development and evaluation of rK28-based point-of-care tests for improving rapid diagnosis of visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75-85%, specificity of 70-92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms. METHODOLOGY/PRINCIPAL FINDINGS: Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83-99.61%; K39: 84.30-98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53-98.95%; K39: 85.54-96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46-99.1 in Sudan and 98.1% [95% CI = 89.93-99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25-93.23%] in Sudan and 88.7% [95% CI = 76.97-95.73%] in Bangladesh). CONCLUSIONS/SIGNIFICANCE: Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care.

Applying TLR synergy in immunotherapy: implications in cutaneous leishmaniasis.

Therapy of intracellular pathogens can be complicated by drug toxicity, drug resistance, and the need for prolonged treatment regimens. One approach that has shown promise is immunotherapy. Leishmaniasis, a vector-borne disease ranked among the six most important tropical infectious diseases by the World Health Organization, has been treated clinically with crude or defined vaccine preparations or cytokines, such as IFN-gamma and GM-CSF, in combination with chemotherapy. We have attempted to develop an improved and defined immunotherapeutic using a mouse model of cutaneous leishmaniasis. We hypothesized that immunotherapy may be improved by using TLR synergy to enhance the parasite-specific immune response. We formulated L110f, a well-established Leishmania poly-protein vaccine candidate, in conjunction with either monophosphoryl lipid A, a TLR4 agonist, or CpG, a TLR9 agonist, or a combination of these, and evaluated anti-Leishmania immune responses in absence or presence of active disease. Only mice treated with L110f plus monophosphoryl lipid A-CpG were able to induce a strong effective T cell response during disease and subsequently cured lesions and reduced parasite burden when compared with mice treated with L110f and either single adjuvant. Our data help to define a correlate of protection during active infection and indicate TLR synergy to be a potentially valuable tool in treating intracellular infections.

Genetic interactions between polymorphisms that affect gene expression in yeast.

Interactions between polymorphisms at different quantitative trait loci (QTLs) are thought to contribute to the genetics of many traits, and can markedly affect the power of genetic studies to detect QTLs. Interacting loci have been identified in many organisms. However, the prevalence of interactions, and the nucleotide changes underlying them, are largely unknown. Here we search for naturally occurring genetic interactions in a large set of quantitative phenotypes--the levels of all transcripts in a cross between two strains of Saccharomyces cerevisiae. For each transcript, we searched for secondary loci interacting with primary QTLs detected by their individual effects. Such locus pairs were estimated to be involved in the inheritance of 57% of transcripts; statistically significant pairs were identified for 225 transcripts. Among these, 67% of secondary loci had individual effects too small to be significant in a genome-wide scan. Engineered polymorphisms in isogenic strains confirmed an interaction between the mating-type locus MAT and the pheromone response gene GPA1. Our results indicate that genetic interactions are widespread in the genetics of transcript levels, and that many QTLs will be missed by single-locus tests but can be detected by two-stage tests that allow for interactions.

Local regulatory variation in Saccharomyces cerevisiae.

Naturally occurring sequence variation that affects gene expression is an important source of phenotypic differences among individuals within a species. We and others have previously shown that such regulatory variation can occur both at the same locus as the gene whose expression it affects (local regulatory variation) and elsewhere in the genome at trans-acting factors. Here we present a detailed analysis of genome-wide local regulatory variation in Saccharomyces cerevisiae. We used genetic linkage analysis to show that nearly a quarter of all yeast genes contain local regulatory variation between two divergent strains. We measured allele-specific expression in a diploid hybrid of the two strains for 77 genes showing strong self-linkage and found that in 52%-78% of these genes, local regulatory variation acts directly in cis. We also experimentally confirmed one example in which local regulatory variation in the gene AMN1 acts in trans through a feedback loop. Genome-wide sequence analysis revealed that genes subject to local regulatory variation show increased polymorphism in the promoter regions, and that some but not all of this increase is due to polymorphisms in predicted transcription factor binding sites. Increased polymorphism was also found in the 3' untranslated regions of these genes. These findings point to the importance of cis-acting variation, but also suggest that there is a diverse set of mechanisms through which local variation can affect gene expression levels.

Simultaneous genotyping, gene-expression measurement, and detection of allele-specific expression with oligonucleotide arrays.

Oligonucleotide microarrays provide a high-throughput method for exploring genomes. In addition to their utility for gene-expression analysis, oligonucleotide-expression arrays have also been used to perform genotyping on genomic DNA. Here, we show that in segregants from a cross between two unrelated strains of Saccharomyces cerevisiae, high-quality genotype data can also be obtained when mRNA is hybridized to an oligonucleotide-expression array. We were able to identify and genotype nearly 1000 polymorphisms at an error rate close to 3% in segregants and at an error rate of 7% in diploid strains, a performance comparable to methods using genomic DNA. In addition, we demonstrate how simultaneous genotyping and gene-expression profiling can reveal cis-regulatory variation by screening hundreds of genes for allele-specific expression. With this method, we discovered 70 ORFs with evidence for preferential expression of one allele in a diploid hybrid of two S. cerevisiae strains.

Trans-acting regulatory variation in Saccharomyces cerevisiae and the role of transcription factors.

Natural genetic variation can cause significant differences in gene expression, but little is known about the polymorphisms that affect gene regulation. We analyzed regulatory variation in a cross between laboratory and wild strains of Saccharomyces cerevisiae. Clustering and linkage analysis defined groups of coregulated genes and the loci involved in their regulation. Most expression differences mapped to trans-acting loci. Positional cloning and functional assays showed that polymorphisms in GPA1 and AMN1 affect expression of genes involved in pheromone response and daughter cell separation, respectively. We also asked whether particular classes of genes were more likely to contain trans-regulatory polymorphisms. Notably, transcription factors showed no enrichment, and trans-regulatory variation seems to be broadly dispersed across classes of genes with different molecular functions.