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Genetic characterization of Lassa virus strains isolated from 2012 to 2016 in southeastern Nigeria

30 November 2018 - 10:00pm

by Olamide K. Oloniniyi, Uche S. Unigwe, Sayaka Okada, Mayuko Kimura, Shota Koyano, Yukiko Miyazaki, Michael O. Iroezindu, Nnenna A. Ajayi, Chinedu M. Chukwubike, Nneka M. Chika-Igwenyi, Anne C. Ndu, Damian U. Nwidi, Haruka Abe, Shuzo Urata, Yohei Kurosaki, Jiro Yasuda

Lassa virus (LASV) is endemic in parts of West Africa where it causes Lassa fever (LF), a viral hemorrhagic fever with frequent fatal outcomes. The diverse LASV strains are grouped into six major lineages based on the geographical location of the isolated strains. In this study, we have focused on the lineage II strains from southern Nigeria. We determined the viral sequences from positive cases of LF reported at tertiary hospitals in Ebonyi and Enugu between 2012 and 2016. Reverse transcription-polymerase chain reaction (RT-PCR) showed that 29 out of 123 suspected cases were positive for the virus among which 11 viral gene sequences were determined. Phylogenetic analysis of the complete coding sequences of the four viral proteins revealed that lineage II strains are broadly divided into two genetic clades that diverged from a common ancestor 195 years ago. One clade, consisting of strains from Ebonyi and Enugu, was more conserved than the other from Irrua, although the four viral proteins were evolving at similar rates in both clades. These results suggested that the viruses of these clades have been distinctively evolving in geographically separate parts of southern Nigeria. Furthermore, the epidemiological data of the 2014 outbreak highlighted the role of human-to-human transmission in this outbreak, which was supported by phylogenetic analysis showing that 13 of the 16 sequences clustered together. These results provide new insights into the evolution of LASV in southern Nigeria and have important implications for vaccine development, diagnostic assay design, and LF outbreak management.

Molecular genotyping, diversity studies and high-resolution molecular markers unveiled by microsatellites in <i>Giardia duodenalis</i>

30 November 2018 - 10:00pm

by Maurício Durigan, Claudio Benício Cardoso-Silva, Maísa Ciampi-Guillardi, Guilherme Toledo-Silva, Gustavo M. Mori, Regina M. B. Franco, Anete P. Souza

Background

Giardia duodenalis (synonyms G. lamblia and G. intestinalis) is an enteric protozoan parasite of a wide range of mammalian hosts, including humans and various domestic and wild animals. There is considerable genetic variability in G. duodenalis and isolates of this parasite have been divided into eight genetic assemblages. Microsatellites markers can be used to discriminate isolates with a high level of sensitivity. This study was conducted to identify and characterize genomic microsatellites (simple sequence repeats—SSRs), sequences of one- to six-nucleotide motifs repeated in tandem, present in the available genomes of G. duodenalis and to develop new markers that can serve as a tool for detection and for characterizing the genetic diversity of this parasite.

Methodology/ Principal findings

For each genetic assemblage, polymorphism levels for the microsatellite markers were evaluated. After performing the analysis using the MISA and SciRoKo software, 1,853 simple sequence repeats (SSRs) were identified. In all the genomes, trinucleotide repeats were the most common class followed by tetranucleotide. Many of the SSR loci are assemblage-specific, and 36 SSR loci shared among all the genomes were identified. Together with hypothetical proteins, variant-specific surface proteins represented nearly half of the annotated SSR loci. The results regarding the most common repeat among the SSRs led us to infer that positive selection occurred to avoid frameshift mutations. Additionally, based on inter- and intra-genetic assemblages polymorphism analyses, we unveiled previously undetected genetic variation, indicating that the microsatellite markers we developed are useful molecular tools for epidemiological inferences based on population genetics patterns and processes.

Conclusions

There is increasing demand for the development of new molecular markers and for the characterization of pathogens at a higher resolution level. In this study, we present 60 G. duodenalis microsatellites markers that exhibited high polymerase chain reaction (PCR) amplification efficiency among the different genetic assemblages. Twenty of these markers presented nucleotide sequence polymorphisms and may be used as a genotyping tool. The monomorphic markers can be used for the detection of the parasite at the species and genetic assemblage level. These polymorphic markers revealed a genetic diversity that was previously undetectable, thus they can be considered valuable molecular tools for high resolution markers in future studies investigating Giardia and may also be used for epidemiological inferences based on populations genetics patterns and processes.

Laboratory challenges of Plasmodium species identification in Aceh Province, Indonesia, a malaria elimination setting with newly discovered <i>P</i>. <i>knowlesi</i>

30 November 2018 - 10:00pm

by Farah N. Coutrier, Yusrifar K. Tirta, Chris Cotter, Iska Zarlinda, Iveth J. González, Alanna Schwartz, Cut Maneh, Jutta Marfurt, Maxwell Murphy, Herdiana Herdiana, Nicholas M. Anstey, Bryan Greenhouse, Michelle S. Hsiang, Rintis Noviyanti

The discovery of the life-threatening zoonotic infection Plasmodium knowlesi has added to the challenges of prompt and accurate malaria diagnosis and surveillance. In this study from Aceh Province, Indonesia, a malaria elimination setting where P. knowlesi endemicity was not previously known, we report the laboratory investigation and difficulties encountered when using molecular detection methods for quality assurance of microscopically identified clinical cases. From 2014 to 2015, 20 (49%) P. falciparum, 16 (39%) P. vivax, 3 (7%) P. malariae, and 2 (5%) indeterminate species were identified by microscopy from four sentinel health facilities. At a provincial-level reference laboratory, loop-mediated isothermal amplification (LAMP), a field-friendly molecular method, was performed and confirmed Plasmodium in all samples though further species-identification was limited by the unavailability of non-falciparum species-specific testing with the platform used. At a national reference laboratory, several molecular methods including nested PCR (nPCR) targeting the 18 small sub-unit (18S) ribosomal RNA, nPCR targeting the cytochrome-b (cytb) gene, a P. knowlesi-specific nPCR, and finally sequencing, were necessary to ultimately classify the samples as: 19 (46%) P. knowlesi, 8 (20%) P. falciparum, 14 (34%) P. vivax. Microscopy was unable to identify or mis-classified up to 56% of confirmed cases, including all cases of P. knowlesi. With the nPCR methods targeting the four human-only species, P. knowlesi was missed (18S rRNA method) or showed cross-reactivity for P. vivax (cytb method). To facilitate diagnosis and management of potentially fatal P. knowlesi infection and surveillance for elimination of human-only malaria in Indonesia and other affected settings, new detection methods are needed for testing at the point-of-care and in local reference laboratories.

The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from <i>Echinococcus granulosus sensu lato</i> and protect mice against secondary cystic echinococcosis

30 November 2018 - 10:00pm

by Gustavo Mourglia-Ettlin, Sebastián Miles, María Velasco-De-Andrés, Noelia Armiger-Borràs, Marcela Cucher, Sylvia Dematteis, Francisco Lozano

Background

Scavenger Receptors (SRs) from the host’s innate immune system are known to bind multiple ligands to promote the removal of non-self or altered-self targets. CD5 and CD6 are two highly homologous class I SRs mainly expressed on all T cells and the B1a cell subset, and involved in the fine tuning of activation and differentiation signals delivered by the antigen-specific receptors (TCR and BCR, respectively), to which they physically associate. Additionally, CD5 and CD6 have been shown to interact with and sense the presence of conserved pathogen-associated structures from bacteria, fungi and/or viruses.

Methodology/Principal findings

We report herein the interaction of CD5 and CD6 lymphocyte surface receptors with Echinococcus granulosus sensu lato (s.l.). Binding studies show that both soluble and membrane-bound forms of CD5 and CD6 bind to intact viable protoscoleces from E. granulosus s.l. through recognition of metaperiodate-resistant tegumental components. Proteomic analyses allowed identification of thioredoxin peroxidase for CD5, and peptidyl-prolyl cis-trans isomerase (cyclophilin) and endophilin B1 (antigen P-29) for CD6, as their potential interactors. Further in vitro assays demonstrate that membrane-bound or soluble CD5 and CD6 forms differentially modulate the pro- and anti-inflammatory cytokine release induced following peritoneal cells exposure to E. granulosus s.l. tegumental components. Importantly, prophylactic infusion of soluble CD5 or CD6 significantly ameliorated the infection outcome in the mouse model of secondary cystic echinococcosis.

Conclusions/Significance

Taken together, the results expand the pathogen binding properties of CD5 and CD6 and provide novel evidence for their therapeutic potential in human cystic echinococcosis.

Virulotyping of <i>Salmonella enterica</i> serovar Typhi isolates from Pakistan: Absence of complete SPI-10 in Vi negative isolates

30 November 2018 - 10:00pm

by Sadia Liaquat, Yasra Sarwar, Aamir Ali, Abdul Haque, Muhammad Farooq, Ilargi Martinez-Ballesteros, Lorena Laorden, Javier Garaizar, Joseba Bikandi

The pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, is mainly attributed to the acquisition of horizontally acquired DNA elements. Salmonella pathogenicity islands (SPIs) are indubitably the most important form of horizontally acquired DNA with respect to pathogenesis of this bacterium. The insertion or deletion of any of these transferrable SPIs may have impact on the virulence potential of S. Typhi. In this study, the virulence potential and genetic relatedness of 35 S. Typhi isolates, collected from 2004 to 2013 was determined by identification of SPI and non-SPI virulence factors through a combination of techniques including virulotyping, Whole Genome Sequencing (WGS), and Variable Number of Tandem Repeats (VNTR) profiling. In order to determine the virulence potential of local S. Typhi isolates, 56 virulence related genes were studied by PCR. These genes are located in the core as well as accessory genome (SPIs and plasmid). Major variations among studied virulence determinants were found in case of SPI-7 and SPI-10 associated genes. On the basis of presence of virulence related genes, the studied S. Typhi isolates from Pakistan were clustered into two virulotypes Vi-positive and Vi-negative. Interestingly, SPI-7 and SPI-10 were collectively absent or present in Vi-negative and Vi-positive strains, respectively. Two Vi-negative and 11 Vi-positive S. Typhi strains were also analyzed by whole genome sequencing (WGS) and their results supported the PCR results. Genetic diversity was tested by VNTR-based molecular typing. All 35 isolates were clustered into five groups. Overall, all Vi-negative isolates were placed in a single group (T5) whereas Vi-positive isolates were grouped into four types. Vi-negative and Vi-positive isolates were mutually exclusive. This is the first report on the comparative distribution of SPI and non-SPI related virulence genes in Vi-negative and Vi-positive S. Typhi isolates with an important finding that SPI-10 is absent in all Vi-negative isolates.

High seroprevalence of Strongyloides stercoralis among individuals from endemic areas considered for solid organ transplant donation: A retrospective serum-bank based study

29 November 2018 - 10:00pm

by Joan Gómez-Junyent, David Paredes, Juan Carlos Hurtado, Ana Requena-Mendez, Angel Ruiz, Maria Eugenia Valls, Jordi Vila, Jose Muñoz

Background

Strongyloides stercoralis is a worldwide disseminated parasitic disease that can be transmitted from solid organ transplant (SOT) donors to recipients. We determined the serological prevalence of S. stercoralis among deceased individuals from endemic areas considered for SOT donation, using our institution’s serum bank.

Methodology

Retrospective study including all deceased potential donors from endemic areas of strongyloidiasis considered for SOT between January 2004 and December 2014 in a tertiary care hospital. The commercial serological test IVD-Elisa was used to determine the serological prevalence of S. stercoralis.

Principal findings

Among 1025 deceased individuals during the study period, 90 were from endemic areas of strongyloidiasis. There were available serum samples for 65 patients and 6 of them tested positive for S. stercoralis (9.23%). Only one of the deceased candidates was finally a donor, without transmitting the infection.

Conclusions

Among deceased individuals from endemic areas considered for SOT donation, seroprevalence of strongyloidiasis was high. This highlights the importance of adhering to current recommendations on screening for S. stercoralis among potential SOT donors at high risk of the infection, together with the need of developing a rapid diagnostic test to fully implement these screening strategies.

Basophils are dispensable for the establishment of protective adaptive immunity against primary and challenge infection with the intestinal helminth parasite <i>Strongyloides ratti</i>

29 November 2018 - 10:00pm

by Martina Reitz, Marie-Luise Brunn, David Voehringer, Minka Breloer

Infections with helminth parasites are controlled by a concerted action of innate and adaptive effector cells in the frame of a type 2 immune response. Basophils are innate effector cells that may also contribute to the initiation and amplification of adaptive immune responses. Here, we use constitutively basophil-deficient Mcpt8-Cre mice to analyze the impact of basophils during initiation and execution of the protective type 2 responses to both, a primary infection and a challenge infection of immune mice with the helminth parasite Strongyloides ratti. Basophil numbers expanded during parasite infection in blood and mesenteric lymph nodes. Basophil deficiency significantly elevated intestinal parasite numbers and fecal release of eggs and larvae during a primary infection. However, basophils were neither required for the initiation of a S. ratti-specific cellular and humoral type 2 immune response nor for the efficient protection against a challenge infection. Production of Th2 cytokines, IgG1 and IgE as well as mast cell activation were not reduced in basophil-deficient Mcpt8-Cre mice compared to basophil-competent Mcpt8-WT littermates. In addition, a challenge infection of immune basophil-deficient and WT mice resulted in a comparable reduction of tissue migrating larvae, parasites in the intestine and fecal release of eggs and L1 compared to mice infected for the first time. We have shown previously that S. ratti infection induced expansion of Foxp3+ regulatory T cells that interfered with efficient parasite expulsion. Here we show that depletion of regulatory T cells reduced intestinal parasite burden also in absence of basophils. Thus basophils were not targeted specifically by S. ratti-mediated immune evasive mechanisms. Our collective data rather suggests that basophils are non-redundant innate effector cells during murine Strongyloides infections that contribute to the early control of intestinal parasite burden.

Integrating evidence, models and maps to enhance Chagas disease vector surveillance

29 November 2018 - 10:00pm

by Alexander Gutfraind, Jennifer K. Peterson, Erica Billig Rose, Claudia Arevalo-Nieto, Justin Sheen, Gian Franco Condori-Luna, Narender Tankasala, Ricardo Castillo-Neyra, Carlos Condori-Pino, Priyanka Anand, Cesar Naquira-Velarde, Michael Z. Levy

Background

Until recently, the Chagas disease vector, Triatoma infestans, was widespread in Arequipa, Perú, but as a result of a decades-long campaign in which over 70,000 houses were treated with insecticides, infestation prevalence is now greatly reduced. To monitor for T. infestans resurgence, the city is currently in a surveillance phase in which a sample of houses is selected for inspection each year. Despite extensive data from the control campaign that could be used to inform surveillance, the selection of houses to inspect is often carried out haphazardly or by convenience. Therefore, we asked, how can we enhance efforts toward preventing T. infestans resurgence by creating the opportunity for vector surveillance to be informed by data?

Methodology/principal findings

To this end, we developed a mobile app that provides vector infestation risk maps generated with data from the control campaign run in a predictive model. The app is intended to enhance vector surveillance activities by giving inspectors the opportunity to incorporate the infestation risk information into their surveillance activities, but it does not dictate which houses to surveil. Therefore, a critical question becomes, will inspectors use the risk information? To answer this question, we ran a pilot study in which we compared surveillance using the app to the current practice (paper maps). We hypothesized that inspectors would use the risk information provided by the app, as measured by the frequency of higher risk houses visited, and qualitative analyses of inspector movement patterns in the field. We also compared the efficiency of both mediums to identify factors that might discourage risk information use. Over the course of ten days (five with each medium), 1,081 houses were visited using the paper maps, of which 366 (34%) were inspected, while 1,038 houses were visited using the app, with 401 (39%) inspected. Five out of eight inspectors (62.5%) visited more higher risk houses when using the app (Fisher’s exact test, p < 0.001). Among all inspectors, there was an upward shift in proportional visits to higher risk houses when using the app (Mantel-Haenszel test, common odds ratio (OR) = 2.42, 95% CI 2.00–2.92), and in a second analysis using generalized linear mixed models, app use increased the odds of visiting a higher risk house 2.73-fold (95% CI 2.24–3.32), suggesting that the risk information provided by the app was used by most inspectors. Qualitative analyses of inspector movement revealed indications of risk information use in seven out of eight (87.5%) inspectors. There was no difference between the app and paper maps in the number of houses visited (paired t-test, p = 0.67) or inspected (p = 0.17), suggesting that app use did not reduce surveillance efficiency.

Conclusions/significance

Without staying vigilant to remaining and re-emerging vector foci following a vector control campaign, disease transmission eventually returns and progress achieved is reversed. Our results suggest that, when provided the opportunity, most inspectors will use risk information to direct their surveillance activities, at least over the short term. The study is an initial, but key, step toward evidence-based vector surveillance.

Implementation science: Epidemiology and feeding profiles of the Chagas vector <i>Triatoma dimidiata</i> prior to Ecohealth intervention for three locations in Central America

28 November 2018 - 10:00pm

by Raquel Asunción Lima-Cordón, Lori Stevens, Elizabeth Solórzano Ortíz, Gabriela Anaité Rodas, Salvador Castellanos, Antonieta Rodas, Vianney Abrego, Concepción Zúniga Valeriano, María Carlota Monroy

The Ecohealth strategy is a multidisciplinary data-driven approach used to improve the quality of people’s lives in Chagas disease endemic areas, such as regions of Central America. Chagas is a vector-borne disease caused by the parasite Trypanosoma cruzi. In Central America, the main vector is Triatoma dimidiata. Because successful implementation of the Ecohealth approach reduced home infestation in Jutiapa department, Guatemala, it was scaled-up to three localities, one in each of three Central American countries (Texistepeque, El Salvador; San Marcos de la Sierra, Honduras and Olopa, Guatemala). As a basis for the house improvement phase of the Ecohealth program, we determined if the localities differ in the role of sylvatic, synanthropic and domestic animals in the Chagas transmission cycle by measuring entomological indices, blood meal sources and parasite infection from vectors collected in and around houses. The Polymerase Chain Reaction (PCR) with taxa specific primers to detect both, blood sources and parasite infection, was used to assess 71 T. dimidiata from Texistepeque, 84 from San Marcos de la Sierra and 568 from Olopa. Our results show that infestation (12.98%) and colonization (8.95%) indices were highest in Olopa; whereas T. cruzi prevalence was higher in Texistepeque and San Marcos de la Sierra (>40%) than Olopa (8%). The blood meal source profiles showed that in Olopa, opossum might be important in linking the sylvatic and domestic Chagas transmission cycle, whereas in San Marcos de la Sierra dogs play a major role in maintaining domestic transmission. For Texistepeque, bird was the major blood meal source followed by human. When examining the different life stages, we found that in Olopa, the proportion bugs infected with T. cruzi is higher in adults than nymphs. These findings highlight the importance of location-based recommendations for decreasing human-vector contact in the control of Chagas disease.

Antibody responses to Zika virus proteins in pregnant and non-pregnant macaques

27 November 2018 - 10:00pm

by Anna S. Heffron, Emma L. Mohr, David Baker, Amelia K. Haj, Connor R. Buechler, Adam Bailey, Dawn M. Dudley, Christina M. Newman, Mariel S. Mohns, Michelle Koenig, Meghan E. Breitbach, Mustafa Rasheed, Laurel M. Stewart, Jens Eickhoff, Richard S. Pinapati, Erica Beckman, Hanying Li, Jigar Patel, John C. Tan, David H. O’Connor

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens.

VDR polymorphism, gene expression and vitamin D levels in leprosy patients from North Indian population

27 November 2018 - 10:00pm

by Itu Singh, Mallika Lavania, Vinay Kumar Pathak, Madhvi Ahuja, Ravindra P. Turankar, Vikram Singh, Utpal Sengupta

Background

Leprosy is a chronic infectious disease caused by Mycobacterium leprae and mainly affects skin, peripheral nerves. Vitamin D receptor (VDR) gene polymorphism has been found to be associated with leprosy. Vitamin D has been shown to control several host immunomodulating properties through VDR gene. Vitamin D deficiency was also found to be linked to an increased risk for several infections and metabolic diseases.

Objective

In the present study, we investigated the association of VDR gene polymorphism, mRNA gene expression of VDR and the vitamin D levels with leprosy and its reactional states.

Methodology

A total of 305 leprosy patients consisting of tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous leprosy (LL), as well as 200 healthy controls were enrolled in the study. We identified single nucleotide polymorphisms (SNPs) of VDR Taq1, Fok1 and Apa1, as well as the expression of VDR mRNA gene using PCR-based restriction fragment length polymorphism (RFLP) analysis and real-time PCR respectively. We also performed ELISA to measure vitamin D levels.

Result

We observed that SNP of VDR gene (Fok1 and Taq1) are associated with the leprosy disease. The allelic frequency distribution of T and t allele (p = 0.0037), F and f allele (p = 0.0024) was significantly higher in leprosy patients and healthy controls. ff genotype of Fok1 was found to be associated with leprosy patients [p = 0.0004; OR (95% CI) 3.148 (1.662–5.965)]. The recessive model of Fok1 genotype was also found to be significantly associated in leprosy patients in comparison to healthy controls [p = 0.00004; OR (95% CI) 2.85 (1.56–5.22)]. Leprosy patients are significantly associated with t-F-a haplotype. Further, VDR gene expression was found to be lower in non-reaction group compared to that of reaction group of leprosy and healthy controls. Paradoxically, we noted no difference in the levels of vitamin D between leprosy patients and healthy controls.

Conclusion

Blood levels of vitamin D do not play any role in clinical manifestations of any forms of leprosy. ff genotype of Fok1 and tt genotype of Taq1 was found to be associated with leprosy per se. Association of t-F-a haplotype with leprosy was found to be significant and could be used as a genetic marker to identify individuals at high risk for developing leprosy. VDR gene expression was lower in TT/BT and BL/LL groups of leprosy in comparison to that of healthy controls.

Insights into antitrypanosomal drug mode-of-action from cytology-based profiling

26 November 2018 - 10:00pm

by James Thomas, Nicola Baker, Sebastian Hutchinson, Caia Dominicus, Anna Trenaman, Lucy Glover, Sam Alsford, David Horn

Chemotherapy continues to have a major impact on reducing the burden of disease caused by trypanosomatids. Unfortunately though, the mode-of-action (MoA) of antitrypanosomal drugs typically remains unclear or only partially characterised. This is the case for four of five current drugs used to treat Human African Trypanosomiasis (HAT); eflornithine is a specific inhibitor of ornithine decarboxylase. Here, we used a panel of T. brucei cellular assays to probe the MoA of the current HAT drugs. The assays included DNA-staining followed by microscopy and quantitative image analysis, or flow cytometry; terminal dUTP nick end labelling to monitor mitochondrial (kinetoplast) DNA replication; antibody-based detection of sites of nuclear DNA damage; and fluorescent dye-staining of mitochondria or lysosomes. We found that melarsoprol inhibited mitosis; nifurtimox reduced mitochondrial protein abundance; pentamidine triggered progressive loss of kinetoplast DNA and disruption of mitochondrial membrane potential; and suramin inhibited cytokinesis. Thus, current antitrypanosomal drugs perturb distinct and specific cellular compartments, structures or cell cycle phases. Further exploiting the findings, we show that putative mitogen-activated protein-kinases contribute to the melarsoprol-induced mitotic defect, reminiscent of the mitotic arrest associated signalling cascade triggered by arsenicals in mammalian cells, used to treat leukaemia. Thus, cytology-based profiling can rapidly yield novel insight into antitrypanosomal drug MoA.

Identification and binding mode of a novel <i>Leishmania</i> Trypanothione reductase inhibitor from high throughput screening

26 November 2018 - 10:00pm

by Lorenzo Turcano, Esther Torrente, Antonino Missineo, Matteo Andreini, Marina Gramiccia, Trentina Di Muccio, Ilaria Genovese, Annarita Fiorillo, Steven Harper, Alberto Bresciani, Gianni Colotti, Andrea Ilari

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts.

Anilinoquinoline based inhibitors of trypanosomatid proliferation

26 November 2018 - 10:00pm

by Lori Ferrins, Amrita Sharma, Sarah M. Thomas, Naimee Mehta, Jessey Erath, Scott Tanghe, Susan E. Leed, Ana Rodriguez, Kojo Mensa-Wilmot, Richard J. Sciotti, Kirsten Gillingwater, Michael P. Pollastri

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.

Aerosol exposure to intermediate size Nipah virus particles induces neurological disease in African green monkeys

21 November 2018 - 10:00pm

by Dima A. Hammoud, Margaret R. Lentz, Abigail Lara, Jordan K. Bohannon, Irwin Feuerstein, Louis Huzella, Peter B. Jahrling, Matthew Lackemeyer, Joseph Laux, Oscar Rojas, Philip Sayre, Jeffrey Solomon, Yu Cong, Vincent Munster, Michael R. Holbrook

Nipah virus (NiV) infection can lead to severe respiratory or neurological disease in humans. Transmission of NiV has been shown to occur through contact with virus contaminated fomites or consumption of contaminated food. Previous results using the African green monkey (AGM) model of NiV infection identified aspects of infection that, while similar to humans, don’t fully recapitulate disease. Previous studies also demonstrate near uniform lethality that is not consistent with human NiV infection. In these studies, aerosol exposure using an intermediate particle size (7μm) was used to mimic potential human exposure by facilitating virus deposition in the upper respiratory tract. Computed tomography evaluation found some animals developed pulmonary parenchymal disease including consolidations, ground-glass opacities, and reactive adenopathy. Despite the lack of neurological signs, magnetic resonance imaging identified distinct brain lesions in three animals, similar to those previously reported in NiV-infected patients. Immunological characterization of tissues collected at necropsy suggested a local pulmonary inflammatory response with increased levels of macrophages in the lung, but a limited neurologic response. These data provide the first clear evidence of neurological involvement in the AGM that recapitulates human disease. With the development of a disease model that is more representative of human disease, these data suggest that NiV infection in the AGM may be appropriate for evaluating therapeutic countermeasures directed at virus-induced neuropathogenesis.

Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes

21 November 2018 - 10:00pm

by Weam I. Zaky, Francesca R. Tomaino, Nils Pilotte, Sandra J. Laney, Steven A. Williams

Background

Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens.

Methodology/Principal findings

Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack.

Conclusions/Significance

Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.

Organization of oversight for integrated control of neglected tropical diseases within Ministries of Health

21 November 2018 - 10:00pm

by Claire Standley, Matthew R. Boyce, Anna Klineberg, Gabrielle Essix, Rebecca Katz

Background

Neglected tropical diseases (NTDs) are communicable diseases that impact approximately 1 billion people, but receive relatively little research, funding, and attention. Many NTDs have similar treatments, epidemiology, and geographic distribution, and as a result, the integration of control efforts can improve accountability, efficiency, and cost-effectiveness of programs. Here, we examine the landscape of efforts towards NTD integration across countries with the highest burden of disease, and review the administrative management of integration in order to identify approaches and pathways for integration.

Methodology and principal findings

We utilized a standardized system to score countries for NTD endemnicity to create a list of 25 countries with the highest overall burden of NTDs. We then conducted a literature review to characterize the NTD control programs in the focus countries. Six countries were selected for key informant interviews to validate literature review results and gather additional data on opportunities and obstacles to NTD integration, from an administrative perspective. The majority of countries included in the study were located in Africa, with the remainder from Asia, North America, and South America. Multiple models and pathways were observed for the integration of NTD programs, in combination with other NTD programs, other diseases, or other health programs. Substantial heterogeneity existed with respect to the NTD control programs, and no country had integrated all of their NTD control efforts into a single program. NTDs that can be treated with preventative chemotherapy were frequently integrated into a single program. Leprosy control was also frequently integrated with those of other communicable diseases, and notably tuberculosis. Barriers to NTD integration may result from internal administrative obstacles or external obstacles.

Conclusions

Although many countries have begun to integrate NTD control efforts, additional work will be required to realize the full benefits of integration in most of the countries examined here. Moving forward, NTD integration efforts must ensure that administrative structures are designed to maximize the potential success of integrated programs and account for existing administrative processes.

EAPB0503: An Imiquimod analog with potent <i>in vitro</i> activity against cutaneous leishmaniasis caused by <i>Leishmania major</i> and <i>Leishmania tropica</i>

21 November 2018 - 10:00pm

by Rana El Hajj, Hanady Bou Youness, Laurence Lachaud, Patrick Bastien, Carine Masquefa, Pierre-Antoine Bonnet, Hiba El Hajj, Ibrahim Khalifeh

Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. Syria is endemic for Leishmania tropica and Leishmania major, causing CL in the Eastern Mediterranean. The large-scale displacement of Syrian refugees exacerbated the spread of CL into neighboring countries. Therapeutic interventions against CL include local, systemic and physical treatments. The high risk for drug-resistance to current treatments stresses the need for new therapies. Imiquimod is an immunomodulatory drug with a tested efficacy against L. major species. Yet, Imiquimod efficacy against L. tropica and the molecular mechanisms dictating its potency are still underexplored. In this study, we characterized the effect of Imiquimod against L. tropica and L. major, and characterized the molecular mechanisms dictating its anti-leishmanial efficacy against both strains. We also investigated the potency and molecular mechanisms of an Imiquimod analog, EAPB0503, against these two strains. We have tested the effect of Imiquimod and EAPB0503 on macrophages infected with either L. major, L. tropica strains, or patient-derived freshly isolated L. tropica parasites. The anti-amastigote activity of either drugs was assessed by quantitative real time PCR (RT-PCR) using kinetoplast specific primers, confocal microscopy using the Glycoprotein 63 (Gp63) Leishmania amastigote antibody or by histology staining. The mechanism of action of either drugs on the canonical nuclear factor kappa- B (NF-κB) pathway was determined by western blot, and confocal microscopy. The immune production of cytokines upon treatment of infected macrophages with either drugs was assessed by ELISA. Both drugs reduced amastigote replication. EAPB0503 proved more potent, particularly on the wild type L. tropica amastigotes. Toll-Like Receptor-7 was upregulated, mainly by Imiquimod, and to a lesser extent by EAPB0503. Both drugs activated the NF-κB canonical pathway triggering an immune response and i-NOS upregulation in infected macrophages. Our findings establish Imiquimod as a strong candidate for treating L. tropica and show the higher potency of its analog EAPB0503 against CL.

Epidemiology of dengue and other arboviruses in a cohort of school children and their families in Yucatan, Mexico: Baseline and first year follow-up

21 November 2018 - 10:00pm

by Diana Patricia Rojas, Gloria Abigail Barrera-Fuentes, Norma Pavia-Ruz, Mariel Salgado-Rodriguez, Azael Che-Mendoza, Pablo Manrique-Saide, Gonzalo M. Vazquez-Prokopec, M. Elizabeth Halloran, Ira M. Longini, Hector Gomez-Dantes

Dengue is the most prevalent mosquito-borne viral disease of humans and is caused by the four serotypes of dengue virus. To estimate the incidence of dengue and other arboviruses, we analyzed the baseline and first year follow-up of a prospective school-based cohort study and their families in three cities in the state of Yucatan, Mexico. Through enhanced surveillance activities, acute febrile illnesses in the participants were detected and yearly blood samples were collected to evaluate dengue infection incidence. A Cox model was fitted to identify hazard ratios of arboviral infections in the first year of follow-up of the cohort. The incidence of dengue symptomatic infections observed during the first year of follow-up (2015–2016) was 3.5 cases per 1,000 person-years (95% CI: 1.9, 5.9). The incidence of dengue infections was 33.9 infections per 1,000 person-years (95% CI: 31.7, 48.0). The majority of dengue infections and seroconversions were observed in the younger age groups (≤ 14 years old). Other arboviruses were circulating in the state of Yucatan during the study period. The incidence of symptomatic chikungunya infections was 8.6 per 1,000 person-years (95% CI: 5.8, 12.3) and the incidence of symptomatic Zika infections was 2.3 per 1,000 person-years (95% CI: 0.9, 4.5). Our model shows that having a dengue infection during the first year of follow-up was significantly associated with being female, living in Ticul or Progreso, and being dengue naïve at baseline. Age was not significantly associated with the outcome, it was confounded by prior immunity to dengue that increases with age. This is the first report of a cohort in Latin America that provides incidence estimates of the three arboviruses co-circulating in all age groups. This study provides important information for understanding the epidemiology of dengue and other arboviruses and better informing public health policies.

Dengue seroprevalence in a cohort of schoolchildren and their siblings in Yucatan, Mexico (2015-2016)

21 November 2018 - 10:00pm

by Norma Pavía-Ruz, Gloria Abigail Barrera-Fuentes, Salha Villanueva-Jorge, Azael Che-Mendoza, Julio César Campuzano-Rincón, Pablo Manrique-Saide, Diana Patricia Rojas, Gonzalo M. Vazquez-Prokopec, M. Elizabeth Halloran, Ira M. Longini, Héctor Gómez-Dantés

Background

The implementation of vector control interventions and potential introduction new tools requires baseline data to evaluate their direct and indirect effects. The objective of the study is to present the seroprevalence of dengue infection in a cohort of children 0 to 15 years old followed during 2015 to 2016, the risk factors and the role of enhanced surveillance strategies in three urban sites (Merida, Ticul and Progreso) in Yucatan, Mexico.

Methods

A cohort of school children and their family members was randomly selected in three urban areas with different demographic, social conditions and levels of transmission. We included results from 1,844 children aged 0 to 15 years. Serum samples were tested for IgG, NS1 and IgM. Enhanced surveillance strategies were established in schools (absenteeism) and cohort families (toll-free number).

Results

Seroprevalence in children 0 to 15 years old was 46.8 (CI 95% 44.1–49.6) with no difference by sex except in Ticul. Prevalence increased with age and was significantly lower in 0 to 5 years old (26.9%, 95% CI:18.4–35.4) compared with 6 to 8 years old (43.9%, 95% CI:40.1–47.7) and 9 to 15 years old (61.4%, 95% CI:58.0–64.8). Sharing the domestic space with other families increased the risk 1.7 times over the individual families that own or rented their house, while risk was significantly higher when kitchen and bathroom were outside. Complete protection with screens in doors and windows decreased risk of infection. Seroprevalence was significantly higher in the medium and high risk areas.

Conclusions

The prevalence of antibodies in children 0 to 15 years in three urban settings in the state of Yucatan describe the high exposure and the heterogenous transmission of dengue virus by risk areas and between schools in the study sites. The enhanced surveillance strategy was useful to improve detection of dengue cases with the coincident transmission of chikungunya and Zika viruses.

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