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by Thaddeus T. Gbem, Mario Waespy, Bettina Hesse, Frank Dietz, Joel Smith, Gloria D. Chechet, Jonathan A. Nok, Sørge KelmTrans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.
Transmission Assessment Surveys (TAS) to Define Endpoints for Lymphatic Filariasis Mass Drug Administration: A Multicenter Evaluation
by Brian K. Chu, Michael Deming, Nana-Kwadwo Biritwum, Windtaré R. Bougma, Améyo M. Dorkenoo, Maged El-Setouhy, Peter U. Fischer, Katherine Gass, Manuel Gonzalez de Peña, Leda Mercado-Hernandez, Dominique Kyelem, Patrick J. Lammie, Rebecca M. Flueckiger, Upendo J. Mwingira, Rahmah Noordin, Irene Offei Owusu, Eric A. Ottesen, Alexandre Pavluck, Nils Pilotte, Ramakrishna U. Rao, Dilhani Samarasekera, Mark A. Schmaedick, Sunil Settinayake, Paul E. Simonsen, Taniawati Supali, Fasihah Taleo, Melissa Torres, Gary J. Weil, Kimberly Y. WonBackground
Lymphatic filariasis (LF) is targeted for global elimination through treatment of entire at-risk populations with repeated annual mass drug administration (MDA). Essential for program success is defining and confirming the appropriate endpoint for MDA when transmission is presumed to have reached a level low enough that it cannot be sustained even in the absence of drug intervention. Guidelines advanced by WHO call for a transmission assessment survey (TAS) to determine if MDA can be stopped within an LF evaluation unit (EU) after at least five effective rounds of annual treatment. To test the value and practicality of these guidelines, a multicenter operational research trial was undertaken in 11 countries covering various geographic and epidemiological settings.Methodology
The TAS was conducted twice in each EU with TAS-1 and TAS-2 approximately 24 months apart. Lot quality assurance sampling (LQAS) formed the basis of the TAS survey design but specific EU characteristics defined the survey site (school or community), eligible population (6–7 year olds or 1st–2nd graders), survey type (systematic or cluster-sampling), target sample size, and critical cutoff (a statistically powered threshold below which transmission is expected to be no longer sustainable). The primary diagnostic tools were the immunochromatographic (ICT) test for W. bancrofti EUs and the BmR1 test (Brugia Rapid or PanLF) for Brugia spp. EUs.Principal Findings/Conclusions
In 10 of 11 EUs, the number of TAS-1 positive cases was below the critical cutoff, indicating that MDA could be stopped. The same results were found in the follow-up TAS-2, therefore, confirming the previous decision outcome. Sample sizes were highly sex and age-representative and closely matched the target value after factoring in estimates of non-participation. The TAS was determined to be a practical and effective evaluation tool for stopping MDA although its validity for longer-term post-MDA surveillance requires further investigation.
Over-Expression of 60s Ribosomal L23a Is Associated with Cellular Proliferation in SAG Resistant Clinical Isolates of Leishmania donovani
by Sanchita Das, Priyanka Shah, Rajendra K. Baharia, Rati Tandon, Prashant Khare, Shyam Sundar, Amogh A. Sahasrabuddhe, M. I. Siddiqi, Anuradha DubeBackground
Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.Methodology and principal findings
The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode.Conclusion/Significance
This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.
Molecular Identification of Nocardia Isolates from Clinical Samples and an Overview of Human Nocardiosis in Brazil
by Paulo Victor Pereira Baio, Juliana Nunes Ramos, Louisy Sanches dos Santos, Morgana Fonseca Soriano, Elisa Martins Ladeira, Mônica Cristina Souza, Thereza Cristina Ferreira Camello, Marcio Garcia Ribeiro, Raphael Hirata Junior, Verônica Viana Vieira, Ana Luíza Mattos-GuaraldiBackground
Nocardia sp. causes a variety of clinical presentations. The incidence of nocardiosis varies geographically according to several factors, such as the prevalence of HIV infections, transplants, neoplastic and rheumatic diseases, as well as climate, socio-economic conditions and laboratory procedures for Nocardia detection and identification. In Brazil the paucity of clinical reports of Nocardia infections suggests that this genus may be underestimated as a cause of human diseases and/or either neglected or misidentified in laboratory specimens. Accurate identification of Nocardia species has become increasingly important for clinical and epidemiological investigations. In this study, seven clinical Nocardia isolates were identified by multilocus sequence analysis (MLSA) and their antimicrobial susceptibility was also determined. Most Nocardia isolates were associated to pulmonary disease.Methodology/Principal Findings
The majority of Brazilian human isolates in cases reported in literature were identified as Nocardia sp. Molecular characterization was used for species identification of Nocardia nova, Nocardia cyriacigeorgica, Nocardia asiatica and Nocardia exalbida/gamkensis. Data indicated that molecular analysis provided a different Nocardia speciation than the initial biochemical identification for most Brazilian isolates. All Nocardia isolates showed susceptibility to trimethoprim-sulfamethoxazole, the antimicrobial of choice in the treatment nocardiosis. N. nova isolated from different clinical specimens from one patient showed identical antimicrobial susceptibility patterns and two distinct clones.Conclusions/Significance
Although Brazil is the world's fifth-largest country in terms of land mass and population, pulmonary, extrapulmonary and systemic forms of nocardiosis were reported in only 6 of the 26 Brazilian states from 1970 to 2013. A least 33.8% of these 46 cases of nocardiosis proved fatal. Interestingly, coinfection by two clones may occur in patients presenting nocardiosis. Nocardia infection may be more common throughout the Brazilian territory and in other developing tropical countries than is currently recognized and MLSA should be used more extensively as an effective method for Nocardia identification.
by Kaye Phillips, Jillian Clare Kohler, Peter Pennefather, Halla Thorsteinsdottir, Joseph WongBackground
This study designed and applied accessible yet systematic methods to generate baseline information about the patterns and structure of Canada's neglected tropical disease (NTD) research network; a network that, until recently, was formed and functioned on the periphery of strategic Canadian research funding.Methodology
Multiple methods were used to conduct this study, including: (1) a systematic bibliometric procedure to capture archival NTD publications and co-authorship data; (2) a country-level “core-periphery” network analysis to measure and map the structure of Canada's NTD co-authorship network including its size, density, cliques, and centralization; and (3) a statistical analysis to test the correlation between the position of countries in Canada's NTD network (“k-core measure”) and the quantity and quality of research produced.Principal Findings
Over the past sixty years (1950–2010), Canadian researchers have contributed to 1,079 NTD publications, specializing in Leishmania, African sleeping sickness, and leprosy. Of this work, 70% of all first authors and co-authors (n = 4,145) have been Canadian. Since the 1990s, however, a network of international co-authorship activity has been emerging, with representation of researchers from 62 different countries; largely researchers from OECD countries (e.g. United States and United Kingdom) and some non-OECD countries (e.g. Brazil and Iran). Canada has a core-periphery NTD international research structure, with a densely connected group of OECD countries and some African nations, such as Uganda and Kenya. Sitting predominantly on the periphery of this research network is a cluster of 16 non-OECD nations that fall within the lowest GDP percentile of the network.Conclusion/Significance
The publication specialties, composition, and position of NTD researchers within Canada's NTD country network provide evidence that while Canadian researchers currently remain the overall gatekeepers of the NTD research they generate; there is opportunity to leverage existing research collaborations and help advance regions and NTD areas that are currently under-developed.
Burden of Mycobacterium ulcerans Disease (Buruli Ulcer) and the Underreporting Ratio in the Territory of Songololo, Democratic Republic of Congo
by Delphin Mavinga Phanzu, Patrick Suykerbuyk, Paul Saunderson, Philippe Ngwala Lukanu, Jean-Bedel Masamba Minuku, Désiré Bofunga B. Imposo, Blanchard Mbadu Diengidi, Makanzu Kayinua, Jean-Jacques Tamfum Muyembe, Pascal Tshindele Lutumba, Bouke C. de Jong, Françoise Portaels, Marleen BoelaertBackground
Cutaneous infection by Mycobacterium ulcerans, also known as Buruli ulcer (BU), represents the third most common mycobacterial disease in the world after tuberculosis and leprosy. Data on the burden of BU disease in the Democratic Republic of Congo are scanty. This study aimed to estimate the prevalence rate and the distribution of BU in the Songololo Territory, and to assess the coverage of the existing hospital-based reporting system.Methods
We conducted a cross-sectional survey (July–August 2008) using the door-to-door method simultaneously in the two rural health zones (RHZ) of the Songololo Territory (RHZ of Kimpese and Nsona-Mpangu), each containing twenty health areas. Cases were defined clinically as active BU and inactive BU in accordance with WHO-case definitions.Results
We detected 775 BU patients (259 active and 516 inactive) in a total population of 237,418 inhabitants. The overall prevalence of BU in Songololo Territory was 3.3/1000 inhabitants, varying from 0 to 27.5/1000 between health areas. Of the 259 patients with active BU, 18 (7%) had been reported in the hospital-based reporting system at Kimpese in the 6–8 months prior to the survey.Conclusion
The survey demonstrated a huge variation of prevalence between health areas in Songololo Territory and gross underreporting of BU cases in the hospital-based reporting system. Data obtained may contribute to better targeted and improved BU control interventions, and serve as a baseline for future assessments of the control program.
De Novo Assembly of a Field Isolate Genome Reveals Novel Plasmodium vivax Erythrocyte Invasion Genes
by James Hester, Ernest R. Chan, Didier Menard, Odile Mercereau-Puijalon, John Barnwell, Peter A. Zimmerman, David SerreRecent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P. vivax reference genome that was generated using DNA from the monkey-adapted strain Salvador I. Any genomic locus deleted in this strain would be lacking in the reference genome sequence and missed in previous analyses. Here, we report de novo assembly of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs, each containing more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P. vivax DNA sequences account for 3.8 million nucleotides and contain 792 predicted genes. Most of these contigs contain members of multigene families and likely originate from telomeric regions. Interestingly, we identify two contigs containing predicted protein coding genes similar to known Plasmodium red blood cell invasion proteins. One gene encodes the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P. knowlesi NBPXb. The second gene harbors all the hallmarks of a Plasmodium erythrocyte-binding protein, including conserved Duffy-binding like and C-terminus cysteine-rich domains. Phylogenetic analysis shows that this novel gene clusters separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showing that this gene is present in most P. vivax genomes and transcribed in blood-stage parasites suggest that P. vivax red blood cell invasion mechanisms may be more complex than currently understood. The strategy employed here complements previous genomic analyses and takes full advantage of next-generation sequencing data to provide a comprehensive characterization of genetic variations in this important malaria parasite. Further analyses of the novel protein coding genes discovered through de novo assembly have the potential to identify genes that influence key aspects of P. vivax biology, including alternative mechanisms of human erythrocyte invasion.
Elimination and Eradication of Neglected Tropical Diseases with Mass Drug Administrations: A Survey of Experts
by Jeremy D. Keenan, Peter J. Hotez, Abdou Amza, Nicole E. Stoller, Bruce D. Gaynor, Travis C. Porco, Thomas M. LietmanBackground
Lymphatic filariasis, onchocerciasis, schistosomiasis, soil-transmitted helminths, and trachoma are the five most prevalent neglected tropical diseases in the world, and each is frequently treated with mass drug administrations. We performed a survey of neglected tropical diseases experts to elicit their opinions on the role of mass drug administrations for the elimination of these infections.Methodology/Principal Findings
We sent an online survey to corresponding authors who had published an article about a neglected tropical disease from 2007 to 2011. Of 825 unique authors who were invited to complete the survey, 365 (44.2%) responded, including 234 (28.4%) who answered questions regarding one of the five most prevalent neglected tropical diseases. Respondents had varying opinions about the goals of programmatic activities for their chosen neglected tropical disease, with elimination or eradication identified as the most important goal by 87% of lymphatic filariasis respondents, 66% of onchocerciasis respondents, 55% of trachoma respondents, 24% of schistosomiasis respondents, and 21% of soil-transmitted helminth respondents. Mass drug administrations, other non-medication health measures, and education were generally thought to be more important for elimination than vector control, development of a new tool, or the presence of a secular trend. Drug resistance was thought to be a major limitation of mass drug administrations for all five neglected tropical diseases. Over half of respondents for lymphatic filariasis and trachoma thought that repeated mass drug administrations could eliminate infection within ten years of the initiation of mass treatments.Conclusions/Significance
Respondents for lymphatic filariasis, onchocerciasis, and trachoma were more enthusiastic about the prospects of elimination and eradication than were respondents for schistosomiasis or soil-transmitted helminths. Mass drug administrations were generally believed to be among the most important factors for the success of elimination efforts for each of the five neglected tropical diseases, highlighting the opportunity for integrating drug distributions.
Estimating Leptospirosis Incidence Using Hospital-Based Surveillance and a Population-Based Health Care Utilization Survey in Tanzania
by Holly M. Biggs, Julian T. Hertz, O. Michael Munishi, Renee L. Galloway, Florian Marks, Wilbrod Saganda, Venance P. Maro, John A. CrumpBackground
The incidence of leptospirosis, a neglected zoonotic disease, is uncertain in Tanzania and much of sub-Saharan Africa, resulting in scarce data on which to prioritize resources for public health interventions and disease control. In this study, we estimate the incidence of leptospirosis in two districts in the Kilimanjaro Region of Tanzania.Methodology/Principal Findings
We conducted a population-based household health care utilization survey in two districts in the Kilimanjaro Region of Tanzania and identified leptospirosis cases at two hospital-based fever sentinel surveillance sites in the Kilimanjaro Region. We used multipliers derived from the health care utilization survey and case numbers from hospital-based surveillance to calculate the incidence of leptospirosis. A total of 810 households were enrolled in the health care utilization survey and multipliers were derived based on responses to questions about health care seeking in the event of febrile illness. Of patients enrolled in fever surveillance over a 1 year period and residing in the 2 districts, 42 (7.14%) of 588 met the case definition for confirmed or probable leptospirosis. After applying multipliers to account for hospital selection, test sensitivity, and study enrollment, we estimated the overall incidence of leptospirosis ranges from 75–102 cases per 100,000 persons annually.Conclusions/Significance
We calculated a high incidence of leptospirosis in two districts in the Kilimanjaro Region of Tanzania, where leptospirosis incidence was previously unknown. Multiplier methods, such as used in this study, may be a feasible method of improving availability of incidence estimates for neglected diseases, such as leptospirosis, in resource constrained settings.
Characteristics of the Human Host Have Little Influence on Which Local Schistosoma mansoni Populations Are Acquired
by Lúcio M. Barbosa, Luciano K. Silva, Eliana A. Reis, Theomira M. Azevedo, Jackson M. Costa, Walter A. Blank, Mitermayer G. Reis, Ronald E. BlantonBackground
Brazil remains the country in the Americas with the highest prevalence of schistosomiasis. A combination of control efforts and development, however, has sharply reduced its intensity and distribution. The acquisition of specific schistosome populations may be dependent on host characteristics such as sex, age, geography, work, habits and culture. How these and other host characteristics align with parasite subpopulations may guide approaches to improve control.Methodology
A cohort of more than 90% of the residents in two rural communities in Brazil participated in an epidemiologic survey of demographic, socio-economic and behavioral characteristics. The variables sex, age, intensity of infection, socio-economic index, % lifetime spent on site, previous infection, and trips outside the district were used to group parasites infecting individuals. Schistosoma mansoni infection status was determined by examination of stools submitted on 3 different days. The aggregate of eggs collected from the whole stool was used to determine degree of population differentiation from allele frequencies for 15 microsatellites.Conclusions/Significance
Infection prevalence was 41% for these communities, and the epidemiologic characteristics were similar to many of the endemic areas of Brazil and the world. Parasite population structuring was observed between the two communities (Jost's D 0.046, CI95% 0.042–0.051), although separated by only 8 km and connected by a highway. No structuring was observed when infected individuals were stratified by host's biologic, demographic or epidemiologic characteristics. Those most heavily infected best reflected the communities' overall parasite diversity. The lack of differentiation within villages suggests that individuals are likely to get infected at the same sites or that the same parasite multilocus genotypes can be found at most sites. The geographic structuring between villages and the lack of structuring by age of the host further supports the impression of a population little affected by migration or drift.
by Inês Loureiro, Joana Faria, Christine Clayton, Sandra Macedo Ribeiro, Nilanjan Roy, Nuno Santarém, Joana Tavares, Anabela Cordeiro-da-SilvaAsparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.
by Ewert Linder, Anne Grote, Sami Varjo, Nina Linder, Marianne Lebbad, Mikael Lundin, Vinod Diwan, Jari Hannuksela, Johan LundinBackground
Microscopy, being relatively easy to perform at low cost, is the universal diagnostic method for detection of most globally important parasitic infections. As quality control is hard to maintain, misdiagnosis is common, which affects both estimates of parasite burdens and patient care. Novel techniques for high-resolution imaging and image transfer over data networks may offer solutions to these problems through provision of education, quality assurance and diagnostics. Imaging can be done directly on image sensor chips, a technique possible to exploit commercially for the development of inexpensive “mini-microscopes”. Images can be transferred for analysis both visually and by computer vision both at point-of-care and at remote locations.Methods/Principal Findings
Here we describe imaging of helminth eggs using mini-microscopes constructed from webcams and mobile phone cameras. The results show that an inexpensive webcam, stripped off its optics to allow direct application of the test sample on the exposed surface of the sensor, yields images of Schistosoma haematobium eggs, which can be identified visually. Using a highly specific image pattern recognition algorithm, 4 out of 5 eggs observed visually could be identified.Conclusions/Significance
As proof of concept we show that an inexpensive imaging device, such as a webcam, may be easily modified into a microscope, for the detection of helminth eggs based on on-chip imaging. Furthermore, algorithms for helminth egg detection by machine vision can be generated for automated diagnostics. The results can be exploited for constructing simple imaging devices for low-cost diagnostics of urogenital schistosomiasis and other neglected tropical infectious diseases.
by Godfrey Bwire, Mugagga Malimbo, Brian Maskery, Young Eun Kim, Vittal Mogasale, Ann LevinIntroduction
In 2010, the World Health Organization released a new cholera vaccine position paper, which recommended the use of cholera vaccines in high-risk endemic areas. However, there is a paucity of data on the burden of cholera in endemic countries. This article reviewed available cholera surveillance data from Uganda and assessed the sufficiency of these data to inform country-specific strategies for cholera vaccination.Methods
The Uganda Ministry of Health conducts cholera surveillance to guide cholera outbreak control activities. This includes reporting the number of cases based on a standardized clinical definition plus systematic laboratory testing of stool samples from suspected cases at the outset and conclusion of outbreaks. This retrospective study analyzes available data by district and by age to estimate incidence rates. Since surveillance activities focus on more severe hospitalized cases and deaths, a sensitivity analysis was conducted to estimate the number of non-severe cases and unrecognized deaths that may not have been captured.Results
Cholera affected all ages, but the geographic distribution of the disease was very heterogeneous in Uganda. We estimated that an average of about 11,000 cholera cases occurred in Uganda each year, which led to approximately 61–182 deaths. The majority of these cases (81%) occurred in a relatively small number of districts comprising just 24% of Uganda's total population. These districts included rural areas bordering the Democratic Republic of Congo, South Sudan, and Kenya as well as the slums of Kampala city. When outbreaks occurred, the average duration was about 15 weeks with a range of 4–44 weeks.Discussion
There is a clear subdivision between high-risk and low-risk districts in Uganda. Vaccination efforts should be focused on the high-risk population. However, enhanced or sentinel surveillance activities should be undertaken to better quantify the endemic disease burden and high-risk populations prior to introducing the vaccine.
by Yordanos B. Molla, Jennifer S. Le Blond, Nicola Wardrop, Peter Baxter, Peter M. Atkinson, Melanie J. Newport, Gail DaveyBackground
Podoconiosis is a non-filarial form of elephantiasis resulting in lymphedema of the lower legs. Previous studies have suggested that podoconiosis arises from the interplay of individual and environmental factors. Here, our aim was to understand the individual-level correlates of podoconiosis by comparing 460 podoconiosis-affected individuals and 707 unaffected controls.Methods/principal findings
This was a case-control study carried out in six kebeles (the lowest governmental administrative unit) in northern Ethiopia. Each kebele was classified into one of three endemicity levels: ‘low’ (prevalence <1%), ‘medium’ (1–5%) and ‘high’ (>5%). A total of 142 (30.7%) households had two or more cases of podoconiosis. Compared to controls, the majority of the cases, especially women, were less educated (OR = 1.7, 95% CI = 1.3 to 2.2), were unmarried (OR = 3.4, 95% CI = 2.6–4.6) and had lower income (t = −4.4, p<0.0001). On average, cases started wearing shoes ten years later than controls. Among cases, age of first wearing shoes was positively correlated with age of onset of podoconiosis (r = 0.6, t = 12.5, p<0.0001). Among all study participants average duration of shoe wearing was less than 30 years. Between both cases and controls, people in ‘high’ and ‘medium’ endemicity kebeles were less likely than people in ‘low’ endemicity areas to ‘ever’ have owned shoes (OR = 0.5, 95% CI = 0.4–0.7).Conclusions
Late use of shoes, usually after the onset of podoconiosis, and inequalities in education, income and marriage were found among cases, particularly among females. There were clustering of cases within households, thus interventions against podoconiosis will benefit from household-targeted case tracing. Most importantly, we identified a secular increase in shoe-wearing over recent years, which may give opportunities to promote shoe-wearing without increasing stigma among those at high risk of podoconiosis.
Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance
by Maia A. Rabaa, Cameron P. Simmons, Annette Fox, Mai Quynh Le, Thuy Thi Thu Nguyen, Hai Yen Le, Robert V. Gibbons, Xuyen Thanh Nguyen, Edward C. Holmes, John G. AaskovDengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.
by David Safronetz, Nafomon Sogoba, Job E. Lopez, Ousmane Maiga, Eric Dahlstrom, Marko Zivcec, Friederike Feldmann, Elaine Haddock, Robert J. Fischer, Jennifer M. Anderson, Vincent J. Munster, Luis Branco, Robert Garry, Stephen F. Porcella, Tom G. Schwan, Heinz FeldmannBackground
Lassa fever is an acute viral illness characterized by multi-organ failure and hemorrhagic manifestations. Lassa fever is most frequently diagnosed in Nigeria, Sierra Leone, Liberia, and Guinea, although sporadic cases have been recorded in other West African countries, including Mali. The etiological agent of Lassa fever is Lassa virus (LASV), an Arenavirus which is maintained in nature and frequently transmitted to humans by Mastomys natalensis. The purpose of this study was to better define the geographic distribution of LASV-infected rodents in sub-Saharan Mali.Methodologies/Principal Findings
Small mammals were live-trapped at various locations across Mali for the purpose of identifying potential zoonotic pathogens. Serological and molecular assays were employed and determined LASV infected rodents were exclusively found in the southern Mali near the border of Côte d'Ivoire. Overall, 19.4% of Mastomys natalensis sampled in this region had evidence of LASV infection, with prevalence rates for individual villages ranging from 0 to 52%. Full-length genomic sequences were determined using high throughput sequencing methodologies for LASV isolates generated from tissue samples of rodents collected in four villages and confirmed the phylogenetic clustering of Malian LASV with strain AV.Conclusions/Significance
The risk of human infections with LASV is greatest in villages in southern Mali. Lassa fever should be considered in the differential diagnosis for febrile individuals and appropriate diagnostic techniques need to be established to determine the incidence of infection and disease in these regions.
by Aldo Henrique Tavares, Kelly Grace Magalhães, Raquel Das Neves Almeida, Rafael Correa, Pedro Henrique Burgel, Anamélia Lorenzetti BoccaParacoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1β that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1β in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL-1 receptor, we demonstrated that IL-1β signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus.
The Intestinal Expulsion of the Roundworm Ascaris suum Is Associated with Eosinophils, Intra-Epithelial T Cells and Decreased Intestinal Transit Time
by Dries Masure, Tao Wang, Johnny Vlaminck, Sarah Claerhoudt, Koen Chiers, Wim Van den Broeck, Jimmy Saunders, Jozef Vercruysse, Peter GeldhofAscaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections.
Soulamarin Isolated from Calophyllum brasiliense (Clusiaceae) Induces Plasma Membrane Permeabilization of Trypanosoma cruzi and Mytochondrial Dysfunction
by Alexandre Rea, Andre G. Tempone, Erika G. Pinto, Juliana T. Mesquita, Eliana Rodrigues, Luciana Grus M. Silva, Patricia Sartorelli, João Henrique G. LagoChagas disease is caused by the parasitic protozoan Trypanosoma cruzi. It has high mortality as well as morbidity rates and usually affects the poorer sections of the population. The development of new, less harmful and more effective drugs is a promising research target, since current standard treatments are highly toxic and administered for long periods. Fractioning of methanol (MeOH) extract of the stem bark of Calophyllum brasiliense (Clusiaceae) resulted in the isolation of the coumarin soulamarin, which was characterized by one- and two-dimensional 1H- and 13C NMR spectroscopy as well as ESI mass spectrometry. All data obtained were consistent with a structure of 6-hydroxy-4-propyl-5-(3-hydroxy-2-methyl-1-oxobutyl)-6″,6″-dimethylpyrane-[2″,3″:8,7]-benzopyran-2-one for soulamarin. Colorimetric MTT assays showed that soulamarin induces trypanocidal effects, and is also active against trypomastigotes. Hemolytic activity tests showed that soulamarin is unable to induce any observable damage to erythrocytes (cmax. = 1,300 µM). The lethal action of soulamarin against T. cruzi was investigated by using amino(4-(6-(amino(iminio)methyl)-1H-indol-2-yl)phenyl)methaniminium chloride (SYTOX Green and 1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[4-(chloromethyl)phenyl]-2,3,6,7,12,13,16,17-octahydro-chloride (MitoTracker Red) as fluorimetric probes. With the former, soulamarin showed dose-dependent permeability of the plasma membrane, relative to fully permeable Triton X-100-treated parasites. Spectrofluorimetric and fluorescence microscopy with the latter revealed that soulamarin also induced a strong depolarization (ca. 97%) of the mitochondrial membrane potential. These data demonstrate that the lethal action of soulamarin towards T. cruzi involves damages to the plasma membrane of the parasite and mitochondrial dysfunction without the additional generation of reactive oxygen species, which may have also contributed to the death of the parasites. Considering the unique mitochondrion of T. cruzi, secondary metabolites of plants affecting the bioenergetic system as soulamarin may contribute as scaffolds for the design of novel and selective drug candidates for neglected diseases, mainly Chagas disease.
by Alex P. McLatchie, Hollie Burrell-Saward, Elmarie Myburgh, Michael D. Lewis, Theresa H. Ward, Jeremy C. Mottram, Simon L. Croft, John M. Kelly, Martin C. TaylorBackground
Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.Methodology/Principal findings
Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5′-variant surface glycoprotein untranslated region (UTR) and a downstream 3′-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.Conclusions/Significance
The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.