Survival of the fittest: How Leishmania evades drug therapy.

Cutaneous leishmaniasis caused by L. tropica is common in the Middle East and treatment failure and drug resistance are known to occur. Several genetic mechanisms: aneuploidy, recombination and loss of heterozygosity, single nucleotide polymorphism (SNP) changes, copy number variation (CNV), and mutation of the H locus associated with drug resistance have been described. We studied SNP and CNV patterns in 22 isolates of L. tropica from Afghanistan, Iran and Syria in a geographic, phylogenetic and antimony exposure context. A high SNP frequency was observed in isolates from Syria on chromosome 23, including the H locus, linked to different ancestry at that chromosome segment. Among the isolates from Afghanistan and Iran, an elevated frequency of nonsynonymous SNPs was observed on several chromosomes. Changes in CNV patterns were seen in isolates exposed to drug pressure, especially for the ferric iron reductase gene. Expanded genes were categorised into five functional categories: translational elongation, mitochondrial transmembrane transport, positive regulation of cellular component organisation, response to stimulus and response to hypoxia. No CNV was identified at the H locus, the MAPK1 gene, the APQ1 gene, nor chromosomes 23, 31 or 36 regardless of previous antimonial exposure. In our study, Leishmania tropica had a jump in the nonsynonymous SNP rates at chromosome 23, including the H locus. CNV was observed among isolates exposed to antimonials, especially involving the gene encoding a ferric iron reductase. Several essential genetic coping mechanisms in the cell were enhanced when exposed to antimony, possibly for the survival of the parasite. Our work supports the perspective that Leishmania uses several mechanisms to adapt to environmental changes and drug exposure.

Parasitic Infections in Solid Organ Transplant Recipients

ABSTRACTLeishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal “somy” (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection.

To explore current patterns of diagnosis and treatment, quantify household economic impact and identify household strategies to cover the costs of visceral leishmaniasis (VL) care in rural Bangladesh. Structured interviews with 113 VL patients from 87 households documenting all provider visits and expenditures for health care for VL, and the ways in which the expenditures were covered. Patients paid a median of 7 visits to six different providers before beginning VL treatment. All visited the subdistrict government hospital at least once. While health care, including antileishmanial drug therapy, is officially available free of charge at government facilities, 79% of patients reported making informal payments for provider access, diagnostics and drug administration; only 14% of patients received their full drug course from this source. For the 58% of patients who purchased the full treatment course, drug cost constituted 34% of direct expenditure. Median direct expenditure for one VL patient was US$87 and median income lost was $40; median total expenditure was 1.2 times annual per capita income of our study population. Households employed multiple coping strategies to cover expenditures, most commonly sale or rental of assets (62%) and taking out loans (64%). Visceral leishmaniasis treatment causes a major economic burden in affected families. Control strategies for VL should facilitate timely, affordable diagnosis and treatment of patients to decrease the infection reservoir and to alleviate the economic burden of VL on households.

Genomic instability, the hallmark of cancer, presents with a variety of mutation types, most commonly single nucleotide variations (SNVs) and copy number variations (CNVs), which traditionally have required different methods for identification. It has proven challenging to simultaneously achieve sufficient breadth to detect CNVs and depth to detect SNVs on samples of limited input amount. The objective of this study was to validate a new methodology for detection of SNVs and CNVs in a single assay. We used a massively multiplex PCR/NGS approach combining an SNV panel covering 585 point mutation hotspots in breast cancer (Cosmic) and a CNV panel targeting 28,000 SNPs designed to detect copy number at chromosomes 1, 2, 13, 18, 21, and X, and focal regions 4p16, 5p15, 7q11, 15q, 17p, 22q11, and 22q13. We applied these panels to breast cancer cell lines and fresh frozen (FF) breast tumor samples; the presence of CNVs in circulating cell-free tumor DNA (ctDNA) in the plasma of breast cancer patients was also investigated. The CNV assay methodology was validated using genomic DNA isolated from 96 human samples with known karyotype; sensitivity to single region deletions or duplications was 100% (71/71) and specificity was 100% for normal regions in the same samples. Single-molecule sensitivity for the detection of CNVs was established by analyzing isolated single cells. Performance of the mutation assay was demonstrated with the analysis of 5 matched tumor and normal cell lines, with 24 out of 27 SNVs known to be present in these cell lines detected. The 3 undetected SNVs were determined to be a result of assay design failure. Also, multiple somatic CNVs (median: 13) were detected in all 5 tumor cell lines. Analysis of the normal cell lines found no cancer related SNVs or CNVs. In 32 FF tumor samples, 78.1% (25/32) had SNVs detected; of samples with SNVs, 88% (22/25) had SNVs in TP53 or PIK3CA. Of the same 32 FF breast tumor samples, 96.9% (31/32) showed full or partial CNVs in at least 1 and up to 15 regions; of the 31 samples with detected CNVs, 93.5% had a CNV of either 1q or 17p, two of the three most prevalent breast cancer CNVs (the 16q region was not represented in this panel). Overall, a combination of SNV and CNV testing allowed identification of genetic changes in 100% of the breast tumor samples, a significant improvement in diagnostic yield than using SNV detection alone. Of the 12 breast cancer patients with matched tumor tissue and plasma samples, 83.3% (10/12) had CNVs detected in tissue. The CNVs present in each primary tumor sample were identified in corresponding plasma ctDNA samples (1 stage IIa, 7 stage IIb, and 2 stage III). The ctDNA fractions in these samples ranged from 0.58 to 4.33%; detection required as few as 86 heterozygous SNPs per CNV. Analysis of ctDNA for cancer-associated mutations may allow earlier, safer and more accurate profiling and monitoring of breast cancer. Thus, this targeted PCR approach offers the promise of an assay able to detect both cancer-associated SNVs and CNVs in the same sample with good sensitivity and specificity, and improved detection rates compared to assays that only detect SNVs. Citation Format: Robert J Pelham, Bernhard G Zimmermann, Eser Kirkizlar, Ryan K Swenerton, Bin Hoang, Onur Sakarya, Joshua E Babiarz, Nicholas Wayham, Tudor Constantin, Styrmir Sigurjonsson, Matthew Rabinowitz, Matthew Hill. Detection of single nucleotide variations and copy number variations in breast cancer tissue and ctDNA samples using single-nucleotide polymorphism-targeted massively multiplexed PCR [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-03.

BackgroundIL8RA and IL8RB, encoded by CXCR1 and CXCR2, are receptors for interleukin (IL)-8 and other CXC chemokines involved in chemotaxis and activation of polymorphonuclear neutrophils (PMN). Variants at CXCR1 and CXCR2 have been associated with susceptibility to cutaneous and mucocutaneous leishmaniasis in Brazil. Here we investigate the role of CXCR1/CXCR2 in visceral leishmaniasis (VL) in India.MethodsThree single nucleotide polymorphisms (SNPs) (rs4674259, rs2234671, rs3138060) that tag linkage disequilibrium blocks across CXCR1/CXCR2 were genotyped in primary family-based (313 cases; 176 nuclear families; 836 individuals) and replication (941 cases; 992 controls) samples. Family- and population-based analyses were performed to look for association between CXCR1/CXCR2 variants and VL. Quantitative RT/PCR was used to compare CXCR1/CXCR2 expression in mRNA from paired splenic aspirates taken before and after treatment from 19 VL patients.ResultsFamily-based analysis using FBAT showed association between VL and SNPs CXCR1_rs2234671 (Z-score = 2.935, P = 0.003) and CXCR1_rs3138060 (Z-score = 2.22, P = 0.026), but not with CXCR2_rs4674259. Logistic regression analysis of the case-control data under an additive model of inheritance showed association between VL and SNPs CXCR2_rs4674259 (OR = 1.15, 95%CI = 1.01-1.31, P = 0.027) and CXCR1_rs3138060 (OR = 1.25, 95%CI = 1.02-1.53, P = 0.028), but not with CXCR1_rs2234671. The 3-locus haplotype T_G_C across these SNPs was shown to be the risk haplotype in both family- (TRANSMIT; P = 0.014) and population- (OR = 1.16, P = 0.028) samples (combined P = 0.002). CXCR2, but not CXCR1, expression was down regulated in pre-treatment compared to post-treatment splenic aspirates (P = 0.021).ConclusionsThis well-powered primary and replication genetic study, together with functional analysis of gene expression, implicate CXCR2 in determining outcome of VL in India.

Introduction Leishmaniasis, a neglected protozoan illness caused by kinetoplastid pathogens encompasses three major clinical subtypes: visceral, cutaneous and mucocutaneous leishmaniasis. Pentavalent antimonials (SbV) have long been the preferred treatment worldwide but increased drug resistance, and significant side effects, including cardiotoxicity have limited their use, particularly in visceral leishmaniasis in India. Similarly, other approved alternatives have concerns such as teratogenicity, high cost, and drug resistance. Areas covered This review aims to provide an overview of emerging therapy for leishmaniasis, highlighting the latest advancements in the field and discuss their potential impact on the treatment and prevention of this neglected tropical disease. It also discusses the limitation of current treatments and need for novel approaches to address them effectively. Expert opinion – For almost eight decades, treatment for all forms of leishmaniasis was solely dependent on SbV, despite several drawbacks like long treatment regimens, cardiotoxicity, and drug resistance. In the past 20 years, three drugs with antileishmanial activity were developed for human disease, but their distribution to endemic regions and accessibility for patients remain neglected. We sorely need new antileishmanial drugs, and we present here the emerging targets for developing new antileishmanial compounds that could be brought into the clinics.

A pathogen’s fitness relates to all biological processes that ensure its survival, reproduction, and transmission in specific conditions. These often include the presence of drugs, forcing pathogens to adapt and develop drug resistance in order to survive. The acquisition of a drug-resistant trait usually comes at a cost, making drug-resistant parasites less fit than their wild-type counterparts. This has important implications on the development of drug resistance and on the frequency of treatment failure cases in endemic regions. Treatment failure in patients suffering from leishmaniasis has been observed for most antileishmanials, but could not always be correlated to drug resistance of the infecting parasite. One similitude of both pentavalent antimonial and miltefosine treatment failure, however, relates to changes in parasite fitness. In the specific case of Leishmania donovani, for example, this may contrast with the usual fitness cost observed in natural drug-resistant organisms and highlights parasite fitness as an important contributor to treatment failure in visceral leishmaniasis in the Indian subcontinent. In this final chapter, we will canvass the knowns and the unknowns of Leishmania fitness at different parasite life stages and for different Leishmania species and discuss its relevance for the development and spread of drug resistance and/or treatment failure in the field. We will also propose new research avenues for leishmaniasis drug development and control in the context of current elimination efforts.

IL28B single nucleotide polymorphisms in the treatment of hepatitis C

Interleukin-27R (WSX-1/T-Cell Cytokine Receptor) Gene-Deficient Mice Display Enhanced Resistance to Leishmania donovani Infection but Develop Severe Liver Immunopathology

The leishmaniasis is a complex disease system, caused by the protozoan parasite Leishmania and transmitted to humans by the vector Lutzomyia spp. Since it is listed as a neglected disease according to the World Health Organization, the aim of this study was to determine the current and future niche of cutaneous and visceral leishmaniasis in the Neotropical region. We built the ecological niche model (ENM) of cutaneous (N= 2 910 occurrences) and visceral (N= 851 occurrences) leishmaniasis using MaxEnt algorithm. Nine bioclimatic variables (BIO1, BIO4, BIO5, BIO6, BIO7, BIO12, BIO13, BIO14, BIO15 (downloaded from the Worldclim) and disease occurrences data were used for the construction of ENM for three periods (current, 2050 and 2070) and four climate change scenarios (RCP 2.6, 4.5, 6.0 y 8.5). We analyzed the number of pixels occupied, identity niche, modified niche (stable, loss, and gain) and seasonality. Our analyses indicated the expansion for cutaneous leishmaniasis (CL), a comparison for visceral leishmaniasis (VL). We rejected the null hypothesis of niche identity between CL and VL with Hellinger’s index = 0.91 (0.92-0.98) and Schoener’s Index = 0.67 (0.85-1.00) but with an overlap niche of 56.3 %. The differences between the two leishmaniasis types were detected in relation to RCP scenarios and niche shifts (area gained / loss). Seasonality was more important for CL. We provided a current picture of CL and VL distributions and the predicted distributional changes associated to different climate change scenarios for the Neotropical region. We can anticipate that increasing range is likely although it will depend locally on the future trends in weather seasonality.

Genome-wide Transcriptome Profiling Reveals the Functional Impact of Rare De Novo and Recurrent CNVs in Autism Spectrum Disorders

Understanding the Role of Extracellular Vesicles in Lenalidomide-Resistance Multiple Myeloma

A central question in Leishmania research is why most species cause cutaneous infections but others cause fatal visceral disease. Interestingly, L. donovani causes both visceral and cutaneous leishmaniasis in Sri Lanka. L. donovani clinical isolates were therefore obtained from cutaneous leishmaniasis (CL-SL) and visceral leishmaniasis (VL-SL) patients from Sri Lanka. The CL-SL isolate was severely attenuated compared to the VL-SL isolate for survival in visceral organs in BALB/c mice. Genomic and transcriptomic analysis argue that gene deletions or pseudogenes specific to CL-SL are not responsible for the difference in disease tropism and that single nucleotide polymorphisms (SNPs) and/or gene copy number variations play a major role in altered pathology. This is illustrated through the observations within showing that a decreased copy number of the A2 gene family and a mutation in the ras-like RagC GTPase enzyme in the mTOR pathway contribute to the attenuation of the CL-SL strain in visceral infection. Overall, this research provides a unique perspective on genetic differences associated with diverse pathologies caused by Leishmania infection.

e12113 Background: : IBC is a rare and aggressive subtype of breast cancer. A significant number of IBC patients who achieve pathologic complete response (no residual tumor in breast and axillary nodes, pCR) relapse after NAC. We hypothesized that circulating cell-free DNA (ctDNA) identified in blood before, during, and after NAC would identify novel ctDNA targets. Methods: Plasma ctDNA was extracted from 43 non-metastatic IBC patients (IRB: LAB04-0698) pre, mid, and post-NAC. The Oncomine Pan Cancer ctDNA Assay (ThermoFisher) was used for library preparation, and high throughput next generation sequencing was performed on a GeneStudio S5XL System (ThermoFisher), following manufacturer’s directions. ThermoFisher Ion Reporter 5.10 Software was used to analyze single nucleotide variants (SNVs), and copy number variants (CNVs). Results: Seventeen patients had pre-NAC ctDNA assessments; 7/17 (41%) had PIK3CA SNVs; 5/7 also had MYC or FGFR2 CNVs. Five of 17 (29%) had TP53 SNVs; 2/5 also had FGFR2 CNVs. Ten patients had mid-NAC ctDNA assessments; 9/10 (90%) had PIK3CA SNVs; 5/9 also had FGFR2 CNVs, 2/9 had FGFR2 and FGFR3 CNVs, 2/9 also had TP53 SNVs, 1/9 had FGFR2 and ERB2 CNVs. Thirty-one patients had post-NAC ctDNA assessments; 5/31 (16%) had PIK3CA SNVs; 2/5 had FGFR2 CNVs, 1/5 also had a TP53 mutation and an FGFR2 CNV, 1/5 had FGFR2 CNV, and FGFR3 CNV. Six of 31 (19%) had TP53 SNVs, 1/6 had CCND1 CNVs, no CNVs were detected in 6 patients with TP53 SNVs. Six of 31 (19%) had MAP2K1 SNVs. Three of 31 (10%) had MET SNVs; 1/3 had CCND3 CNVs, no CNVs were detected in 2 patients with MET SNVs. No SNVs or CNVs were detected in 10/31 (32%) of patients post NAC. Conclusions: ctDNA assessments before, during, and after NAC identified novel targets that could be tested in future adjuvant therapies trials in IBC patients who remain at high risk for relapse.