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<i>Leptospira interrogans</i> causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

27 July 2017 - 9:00pm

by Hiromi Sato, Jenifer Coburn

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways including endothelial barrier damage and inflammation, potentially leading to vascular hyperpermeability and severe illness in vivo. This work provides new insights into the pathophysiological mechanisms of Leptospira infection.

Expression of intra- and extracellular granzymes in patients with typhoid fever

27 July 2017 - 9:00pm

by Hanna K. de Jong, Maria Isabel Garcia-Laorden, Arie J. Hoogendijk, Christopher M. Parry, Rapeephan R. Maude, Arjen M. Dondorp, Mohammed Abul Faiz, Tom van der Poll, Willem Joost Wiersinga

Background

Typhoid fever, caused by the intracellular pathogen Salmonella (S.) enterica serovar Typhi, remains a major cause of morbidity and mortality worldwide. Granzymes are serine proteases promoting cytotoxic lymphocytes mediated eradication of intracellular pathogens via the induction of cell death and which can also play a role in inflammation. We aimed to characterize the expression of extracellular and intracellular granzymes in patients with typhoid fever and whether the extracellular levels of granzyme correlated with IFN-γ release.

Methods and principal findings

We analyzed soluble protein levels of extracellular granzyme A and B in healthy volunteers and patients with confirmed S. Typhi infection on admission and day of discharge, and investigated whether this correlated with interferon (IFN)-γ release, a cytokine significantly expressed in typhoid fever. The intracellular expression of granzyme A, B and K in subsets of lymphocytic cells was determined using flow cytometry. Patients demonstrated a marked increase of extracellular granzyme A and B in acute phase plasma and a correlation of both granzymes with IFN-γ release. In patients, lower plasma levels of granzyme B, but not granzyme A, were found at day of discharge compared to admission, indicating an association of granzyme B with stage of disease. Peripheral blood mononuclear cells of typhoid fever patients had a higher percentage of lymphocytic cells expressing intracellular granzyme A and granzyme B, but not granzyme K, compared to controls.

Conclusion

The marked increase observed in extra- and intracellular levels of granzyme expression in patients with typhoid fever, and the correlation with stage of disease, suggests a role for granzymes in the host response to this disease.

Adding tsetse control to medical activities contributes to decreasing transmission of sleeping sickness in the Mandoul focus (Chad)

27 July 2017 - 9:00pm

by Mahamat Hissene Mahamat, Mallaye Peka, Jean-Baptiste Rayaisse, Kat S. Rock, Mahamat Abdelrahim Toko, Justin Darnas, Guihini Mollo Brahim, Ali Bachar Alkatib, Wilfrid Yoni, Inaki Tirados, Fabrice Courtin, Samuel P. C. Brand, Cyrus Nersy, Idriss Oumar Alfaroukh, Steve J. Torr, Mike J. Lehane, Philippe Solano

Background

Gambian sleeping sickness or HAT (human African trypanosomiasis) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by riverine species of tsetse. A global programme aims to eliminate the disease as a public health problem by 2020 and stop transmission by 2030. In the South of Chad, the Mandoul area is a persistent focus of Gambian sleeping sickness where around 100 HAT cases were still diagnosed and treated annually until 2013. Pre-2014, control of HAT relied solely on case detection and treatment, which lead to a gradual decrease in the number of cases of HAT due to annual screening of the population.

Methods

Because of the persistence of transmission and detection of new cases, we assessed whether the addition of vector control to case detection and treatment could further reduce transmission and consequently, reduce annual incidence of HAT in Mandoul. In particular, we investigated the impact of deploying ‘tiny targets’ which attract and kill tsetse. Before tsetse control commenced, a census of the human population was conducted and their settlements mapped. A pre-intervention survey of tsetse distribution and abundance was implemented in November 2013 and 2600 targets were deployed in the riverine habitats of tsetse in early 2014, 2015 and 2016. Impact on tsetse and on the incidence of sleeping sickness was assessed through nine tsetse monitoring surveys and four medical surveys of the human population in 2014 and 2015. Mathematical modelling was used to assess the relative impact of tsetse control on incidence compared to active and passive screening.

Findings

The census indicated that a population of 38674 inhabitants lived in the vicinity of the Mandoul focus. Within this focus in November 2013, the vector is Glossina fuscipes fuscipes and the mean catch of tsetse from traps was 0.7 flies/trap/day (range, 0–26). The catch of tsetse from 44 sentinel biconical traps declined after target deployment with only five tsetse being caught in nine surveys giving a mean catch of 0.005 tsetse/trap/day. Modelling indicates that 70.4% (95% CI: 51–95%) of the reduction in reported cases between 2013 and 2015 can be attributed to vector control with the rest due to medical intervention. Similarly tiny targets are estimated to have reduced new infections dramatically with 62.8% (95% CI: 59–66%) of the reduction due to tsetse control, and 8.5% (95% 8–9%) to enhanced passive detection. Model predictions anticipate that elimination as a public health problem could be achieved by 2018 in this focus if vector control and screening continue at the present level and, furthermore, there may have been virtually no transmission since 2015.

Conclusion

This work shows that tiny targets reduced the numbers of tsetse in this focus in Chad, which may have interrupted transmission and the combination of tsetse control to medical detection and treatment has played a major role in reducing in HAT incidence in 2014 and 2015.

Increased level and interferon-γ production of circulating natural killer cells in patients with scrub typhus

27 July 2017 - 9:00pm

by Seung-Ji Kang, Hye-Mi Jin, Young-Nan Cho, Seong Eun Kim, Uh Jin Kim, Kyung-Hwa Park, Hee-Chang Jang, Sook-In Jung, Seung-Jung Kee, Yong-Wook Park

Background

Natural killer (NK) cells are essential immune cells against several pathogens. Not much is known regarding the roll of NK cells in Orientia tsutsugamushi infection. Thus, this study aims to determine the level, function, and clinical relevance of NK cells in patients with scrub typhus.

Methodology/Principal findings

This study enrolled fifty-six scrub typhus patients and 56 health controls (HCs). The patients were divided into subgroups according to their disease severity. A flow cytometry measured NK cell level and function in peripheral blood. Circulating NK cell levels and CD69 expressions were significantly increased in scrub typhus patients. Increased NK cell levels reflected disease severity. In scrub typhus patients, tests showed their NK cells produced higher amounts of interferon (IFN)-γ after stimulation with interleukin (IL)-12 and IL-18 relative to those of HCs. Meanwhile, between scrub typhus patients and HCs, the cytotoxicity and degranulation of NK cells against K562 were comparable. CD69 expressions were recovered to the normal levels in the remission phase.

Conclusions

This study shows that circulating NK cells are activated and numerically increased, and they produced more IFN-γ in scrub typhus patients.

A novel rapid test for detecting antibody responses to <i>Loa loa</i> infections

27 July 2017 - 9:00pm

by Bijan Pedram, Valérie Pasquetto, Papa M. Drame, Yongchang Ji, Maria J. Gonzalez-Moa, Richard K. Baldwin, Thomas B. Nutman, Marco A. Biamonte

Ivermectin-based mass drug administration (MDA) programs have achieved remarkable success towards the elimination of onchocerciasis and lymphatic filariasis. However, their full implementation has been hindered in Central Africa by the occurrence of ivermectin-related severe adverse events (SAEs) in a subset of individuals with high circulating levels of Loa loa microfilariae. Extending MDA to areas with coincident L. loa infection is problematic, and inexpensive point-of-care tests for L. loa are acutely needed. Herein, we present a lateral flow assay (LFA) to identify subjects with a serological response to Ll-SXP-1, a specific and validated marker of L. loa. The test was evaluated on serum samples from patients infected with L. loa (n = 109) and other helminths (n = 204), as well as on uninfected controls (n = 77). When read with the naked eye, the test was 94% sensitive for L. loa infection and was 100% specific when sera from healthy endemic and non-endemic controls or from those with S. stercoralis infections were used as the comparators. When sera of patients with O. volvulus, W. bancrofti, or M. perstans were used as the comparators, the specificity of the LFA was 82%, 87%, and 88%, respectively. A companion smartphone reader allowed measurement of the test line intensities and establishment of cutoff values. With a cutoff of 600 Units, the assay sensitivity decreased to 71%, but the specificity increased to 96% for O. volvulus, 100% for W. bancrofti, and 100% for M. perstans-infected individuals. The LFA may find applications in refining the current maps of L. loa prevalence, which are needed to eliminate onchocerciasis and lymphatic filariasis from the African continent.

Transmission risk of two chikungunya lineages by invasive mosquito vectors from Florida and the Dominican Republic

27 July 2017 - 9:00pm

by Barry W. Alto, Keenan Wiggins, Bradley Eastmond, Daniel Velez, L. Philip Lounibos, Cynthia C. Lord

Between 2014 and 2016 more than 3,800 imported human cases of chikungunya fever in Florida highlight the high risk for local transmission. To examine the potential for sustained local transmission of chikungunya virus (CHIKV) in Florida we tested whether local populations of Aedes aegypti and Aedes albopictus show differences in susceptibility to infection and transmission to two emergent lineages of CHIKV, Indian Ocean (IOC) and Asian genotypes (AC) in laboratory experiments. All examined populations of Ae. aegypti and Ae. albopictus mosquitoes displayed susceptibility to infection, rapid viral dissemination into the hemocoel, and transmission for both emergent lineages of CHIKV. Aedes albopictus had higher disseminated infection and transmission of IOC sooner after ingesting CHIKV infected blood than Ae. aegypti. Aedes aegypti had higher disseminated infection and transmission later during infection with AC than Ae. albopictus. Viral dissemination and transmission of AC declined during the extrinsic incubation period, suggesting that transmission risk declines with length of infection. Interestingly, the reduction in transmission of AC was less in Ae. aegypti than Ae. albopictus, suggesting that older Ae. aegypti females are relatively more competent vectors than similar aged Ae. albopictus females. Aedes aegypti originating from the Dominican Republic had viral dissemination and transmission rates for IOC and AC strains that were lower than for Florida vectors. We identified small-scale geographic variation in vector competence among Ae. aegypti and Ae. albopictus that may contribute to regional differences in risk of CHIKV transmission in Florida.

Participation of women and children in hunting activities in Sierra Leone and implications for control of zoonotic infections

27 July 2017 - 9:00pm

by Jesse Bonwitt, Martin Kandeh, Michael Dawson, Rashid Ansumana, Foday Sahr, Ann H. Kelly, Hannah Brown

The emergence of infectious diseases of zoonotic origin highlights the need to understand social practices at the animal-human interface. This study provides a qualitative account of interactions between humans and wild animals in predominantly Mende villages of southern Sierra Leone. We conducted fieldwork over 4 months including participant and direct observations, semi-structured interviews (n = 47), spontaneously occurring focus group discussions (n = 12), school essays and informal interviews to describe behaviours that may serve as pathways for zoonotic infection. In this region, hunting is the primary form of contact with wild animals. We describe how these interactions are shaped by socio-cultural contexts, including opportunities to access economic resources and by social obligations and constraints. Our research suggests that the potential for exposure to zoonotic pathogens is more widely distributed across different age, gender and social groups than previously appreciated. We highlight the role of children in hunting, an age group that has previously not been discussed in the context of hunting. The breadth of the "at risk" population forces reconsideration of how we conceptualize, trace and monitor pathogen exposure.

Vertical transmission of human African trypanosomiasis: Clinical evolution and brain MRI of a mother and her son

27 July 2017 - 9:00pm

by Kathleen Gaillot, Marie-Agnès Lauvin, Jean-Philippe Cottier

<i>Aedes ægypti</i> control in urban areas: A systemic approach to a complex dynamic

27 July 2017 - 9:00pm

by Marilia Sá Carvalho, Nildimar Alves Honorio, Leandro Martin Totaro Garcia, Luiz Carlos de Sá Carvalho

Author summary: The available strategy for controlling the diseases transmitted by Aedes ægypti (dengue fever, Zika, and chikungunya) relies on continued community participation. Despite slogans emphasizing how easy it should be, no country has achieved it since the seventies. To better investigate potentially sustainable interventions, we developed a systemic model based on a multidisciplinary approach, integrating as deeply as possible specialized knowledge and field experience. The resulting model is composed of 4 external and 8 internal subsystems and 31 relationships, consistent with the literature and checked over multiple iterations with specialists of the many areas. We analyzed the model and the main feedback loops responsible for the system’s stability, searching for possible interventions that could shift the existing balance. We suggest the introduction of 1 more player, the local primary health care structure, with the potential to change the undesired equilibrium. The health agents in the areas are the first to detect disease cases, and they could stimulate individuals to inform about potential mosquitoes’ breeding sites and bring timely information to the vector-control program. Triggering such an action could introduce changes in people's attitude through a positive feedback loop in the desired direction.

Genome-wide analysis of ivermectin response by <i>Onchocerca volvulus</i> reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

26 July 2017 - 9:00pm

by Stephen R. Doyle, Catherine Bourguinat, Hugues C. Nana-Djeunga, Jonas A. Kengne-Ouafo, Sébastien D. S. Pion, Jean Bopda, Joseph Kamgno, Samuel Wanji, Hua Che, Annette C. Kuesel, Martin Walker, Maria-Gloria Basáñez, Daniel A. Boakye, Mike Y. Osei-Atweneboana, Michel Boussinesq, Roger K. Prichard, Warwick N. Grant

Background

Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana—exposed to more than a decade of regular ivermectin treatment—have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.

Methodology/Principal findings

Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.

Conclusions/Significance

This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations.

Transmission dynamics of co-endemic <i>Plasmodium vivax</i> and <i>P</i>. <i>falciparum</i> in Ethiopia and prevalence of antimalarial resistant genotypes

26 July 2017 - 9:00pm

by Eugenia Lo, Elizabeth Hemming-Schroeder, Delenasaw Yewhalaw, Jennifer Nguyen, Estifanos Kebede, Endalew Zemene, Sisay Getachew, Kora Tushune, Daibin Zhong, Guofa Zhou, Beyene Petros, Guiyun Yan

Ethiopia is one of the few African countries where Plasmodium vivax is co-endemic with P. falciparum. Malaria transmission is seasonal and transmission intensity varies mainly by landscape and climate. Although the recent emergence of drug resistant parasites presents a major issue to malaria control in Ethiopia, little is known about the transmission pathways of parasite species and prevalence of resistant markers. This study used microsatellites to determine population diversity and gene flow patterns of P. falciparum (N = 226) and P. vivax (N = 205), as well as prevalence of drug resistant markers to infer the impact of gene flow and existing malaria treatment regimes. Plasmodium falciparum indicated a higher rate of polyclonal infections than P. vivax. Both species revealed moderate genetic diversity and similar population structure. Populations in the northern highlands were closely related to the eastern Rift Valley, but slightly distinct from the southern basin area. Gene flow via human migrations between the northern and eastern populations were frequent and mostly bidirectional. Landscape genetic analyses indicated that environmental heterogeneity and geographical distance did not constrain parasite gene flow. This may partly explain similar patterns of resistant marker prevalence. In P. falciparum, a high prevalence of mutant alleles was detected in codons related to chloroquine (pfcrt and pfmdr1) and sulfadoxine-pyrimethamine (pfdhps and pfdhfr) resistance. Over 60% of the samples showed pfmdr1 duplications. Nevertheless, no mutation was detected in pfK13 that relates to artemisinin resistance. In P. vivax, while sequences of pvcrt-o were highly conserved and less than 5% of the samples showed pvmdr duplications, over 50% of the samples had pvmdr1 976F mutation. It remains to be tested if this mutation relates to chloroquine resistance. Monitoring the extent of malaria spread and markers of drug resistance is imperative to inform policy for evidence-based antimalarial choice and interventions. To effectively reduce malaria burden in Ethiopia, control efforts should focus on seasonal migrant populations.

The disease burden of human cystic echinococcosis based on HDRs from 2001 to 2014 in Italy

26 July 2017 - 9:00pm

by Toni Piseddu, Diego Brundu, Giovanni Stegel, Federica Loi, Sandro Rolesu, Gabriella Masu, Salvatore Ledda, Giovanna Masala

Background

Cystic echinococcosis (CE) is an important neglected zoonotic parasitic infection belonging to the subgroup of seven Neglected Zoonotic Disease (NZDs) included in the World Health Organization’s official list of 18 Neglected Tropical Diseases (NTDs). CE causes serious global human health concerns and leads to significant economic losses arising from the costs of medical treatment, morbidity, life impairments and fatality rates in human cases. Moreover, CE is endemic in several Italian Regions. The aim of this study is to perform a detailed analysis of the economic burden of hospitalization and treatment costs and to estimate the Disability Adjusted Life Years (DALYs) of CE in Italy.

Methods and findings

In the period from 2001 to 2014, the direct costs of 21,050 Hospital Discharge Records (HDRs) belonging to 12,619 patients with at least one CE-related diagnosis codes were analyzed in order to quantify the economic burden of CE. CE cases average per annum are 901 (min—max = 480–1,583). Direct costs include expenses for hospitalizations, medical and surgical treatment incurred by public and private hospitals and were computed on an individual basis according to Italian Health Ministry legislation. Moreover, we estimated the DALYs for each patient. The Italian financial burden of CE is around € 53 million; the national average economic burden per annum is around € 4 million; the DALYs of the population from 2001 to 2014 are 223.35 annually and 5.26 DALYs per 105 inhabitants.

Conclusion

In Italy, human CE is responsible for significant economic losses in the public health sector. In humans, costs associated with CE have been shown to have a great impact on affected individuals, their families and the community as a whole. This study could be used as a tool to prioritize and make decisions with regard to a surveillance system for this largely preventable yet neglected disease. It demonstrates the need of implementing a CE control program aimed at preventing the considerable economic and social losses it causes in high incidence areas.

Risk of exposure to potential vector mosquitoes for rural workers in Northern Lao PDR

25 July 2017 - 9:00pm

by Julie-Anne A. Tangena, Phoutmany Thammavong, Steve W. Lindsay, Paul T. Brey

Background

One major consequence of economic development in South-East Asia has been a rapid expansion of rubber plantations, in which outbreaks of dengue and malaria have occurred. Here we explored the difference in risk of exposure to potential dengue, Japanese encephalitis (JE), and malaria vectors between rubber workers and those engaged in traditional forest activities in northern Laos PDR.

Methodology/Principal findings

Adult mosquitoes were collected for nine months in secondary forests, mature and immature rubber plantations, and villages. Human behavior data were collected using rapid participatory rural appraisals and surveys. Exposure risk was assessed by combining vector and human behavior and calculating the basic reproduction number (R0) in different typologies. Compared to those that stayed in the village, the risk of dengue vector exposure was higher for those that visited the secondary forests during the day (odds ratio (OR) 36.0), for those living and working in rubber plantations (OR 16.2) and for those that tapped rubber (OR 3.2). Exposure to JE vectors was also higher in the forest (OR 1.4) and, similar when working (OR 1.0) and living in the plantations (OR 0.8). Exposure to malaria vectors was greater in the forest (OR 1.3), similar when working in the plantations (OR 0.9) and lower when living in the plantations (OR 0.6). R0 for dengue was >2.8 for all habitats surveyed, except villages where R0≤0.06. The main malaria vector in all habitats was Anopheles maculatus s.l. in the rainy season and An. minimus s.l. in the dry season.

Conclusions/Significance

The highest risk of exposure to vector mosquitoes occurred when people visit natural forests. However, since rubber workers spend long periods in the rubber plantations, their risk of exposure is increased greatly compared to those who temporarily enter natural forests or remain in the village. This study highlights the necessity of broadening mosquito control to include rubber plantations.

Wetlands, wild Bovidae species richness and sheep density delineate risk of Rift Valley fever outbreaks in the African continent and Arabian Peninsula

25 July 2017 - 9:00pm

by Michael G. Walsh, Allard Willem de Smalen, Siobhan M. Mor

Rift Valley fever (RVF) is an emerging, vector-borne viral zoonosis that has significantly impacted public health, livestock health and production, and food security over the last three decades across large regions of the African continent and the Arabian Peninsula. The potential for expansion of RVF outbreaks within and beyond the range of previous occurrence is unknown. Despite many large national and international epidemics, the landscape epidemiology of RVF remains obscure, particularly with respect to the ecological roles of wildlife reservoirs and surface water features. The current investigation modeled RVF risk throughout Africa and the Arabian Peninsula as a function of a suite of biotic and abiotic landscape features using machine learning methods. Intermittent wetland, wild Bovidae species richness and sheep density were associated with increased landscape suitability to RVF outbreaks. These results suggest the role of wildlife hosts and distinct hydrogeographic landscapes in RVF virus circulation and subsequent outbreaks may be underestimated. These results await validation by studies employing a deeper, field-based interrogation of potential wildlife hosts within high risk taxa.

Tracking the return of <i>Aedes aegypti</i> to Brazil, the major vector of the dengue, chikungunya and Zika viruses

25 July 2017 - 9:00pm

by Panayiota Kotsakiozi, Andrea Gloria-Soria, Adalgisa Caccone, Benjamin Evans, Renata Schama, Ademir Jesus Martins, Jeffrey R. Powell

Background

Aedes aegypti, commonly known as “the yellow fever mosquito”, is of great medical concern today primarily as the major vector of dengue, chikungunya and Zika viruses, although yellow fever remains a serious health concern in some regions. The history of Ae. aegypti in Brazil is of particular interest because the country was subjected to a well-documented eradication program during 1940s-1950s. After cessation of the campaign, the mosquito quickly re-established in the early 1970s with several dengue outbreaks reported during the last 30 years. Brazil can be considered the country suffering the most from the yellow fever mosquito, given the high number of dengue, chikungunya and Zika cases reported in the country, after having once been declared “free of Ae. aegypti”.

Methodology/Principal findings

We used 12 microsatellite markers to infer the genetic structure of Brazilian Ae. aegypti populations, genetic variability, genetic affinities with neighboring geographic areas, and the timing of their arrival and spread. This enabled us to reconstruct their recent history and evaluate whether the reappearance in Brazil was the result of re-invasion from neighboring non-eradicated areas or re-emergence from local refugia surviving the eradication program. Our results indicate a genetic break separating the northern and southern Brazilian Ae. aegypti populations, with further genetic differentiation within each cluster, especially in southern Brazil.

Conclusions/Significance

Based on our results, re-invasions from non-eradicated regions are the most likely scenario for the reappearance of Ae. aegypti in Brazil. While populations in the northern cluster are likely to have descended from Venezuela populations as early as the 1970s, southern populations seem to have derived more recently from northern Brazilian areas. Possible entry points are also revealed within both southern and northern clusters that could inform strategies to control and monitor this important arbovirus vector.

Insecticide resistance is mediated by multiple mechanisms in recently introduced <i>Aedes aegypti</i> from Madeira Island (Portugal)

24 July 2017 - 9:00pm

by Gonçalo Seixas, Linda Grigoraki, David Weetman, José Luís Vicente, Ana Clara Silva, João Pinto, John Vontas, Carla Alexandra Sousa

Background

Aedes aegypti is a major mosquito vector of arboviruses, including dengue, chikungunya and Zika. In 2005, Ae. aegypti was identified for the first time in Madeira Island. Despite an initial insecticide-based vector control program, the species expanded throughout the Southern coast of the island, suggesting the presence of insecticide resistance. Here, we characterized the insecticide resistance status and the underlying mechanisms of two populations of Ae. aegypti from Madeira Island, Funchal and Paúl do Mar.

Methodology/Principal findings

WHO susceptibility bioassays indicated resistance to cyfluthrin, permethrin, fenitrothion and bendiocarb. Use of synergists significantly increased mortality rates, and biochemical assays indicated elevated activities of detoxification enzymes, suggesting the importance of metabolic resistance. Microarray-based transcriptome analysis detected significant upregulation in both populations of nine cytochrome P450 oxidase genes (including four known pyrethroid metabolizing enzymes), the organophosphate metabolizer CCEae3a, Glutathione-S-transferases, and multiple putative cuticle proteins. Genotyping of knockdown resistance loci linked to pyrethroid resistance revealed fixation of the 1534C mutation, and presence with moderate frequencies of the V1016I mutation in each population.

Conclusions/Significance

Significant resistance to three major insecticide classes (pyrethroid, carbamate and organophosphate) is present in Ae. aegypti from Madeira Island, and appears to be mediated by multiple mechanisms. Implementation of appropriate resistance management strategies including rotation of insecticides with alternative modes of action, and methods other than chemical-based vector control are strongly advised to delay or reverse the spread of resistance and achieve efficient control.

Epidemiology of enteroaggregative <i>Escherichia coli</i> infections and associated outcomes in the MAL-ED birth cohort

24 July 2017 - 9:00pm

by Elizabeth T. Rogawski, Richard L. Guerrant, Alexandre Havt, Ila F. N. Lima, Pedro H. Q. S. Medeiros, Jessica C. Seidman, Benjamin J. J. McCormick, Sudhir Babji, Dinesh Hariraju, Ladaporn Bodhidatta, Jasmin Shrestha, Japhat Anania, Athanasia Maro, Amidou Samie, Pablo Peñataro Yori, Shahida Qureshi, Mustafa Mahfuz, Pascal O. Bessong, Margaret N. Kosek, Tahmeed Ahmed, Zulfiqar A. Bhutta, Dennis R. Lang, Michael Gottlieb, Eric R. Houpt, Aldo A. M. Lima, the MAL-ED Network Investigators

Background

Enteroaggregative E. coli (EAEC) have been associated with mildly inflammatory diarrhea in outbreaks and in travelers and have been increasingly recognized as enteric pathogens in young children with and without overt diarrhea. We examined the risk factors for EAEC infections and their associations with environmental enteropathy biomarkers and growth outcomes over the first two years of life in eight low-resource settings of the MAL-ED study.

Methods

EAEC infections were detected by PCR gene probes for aatA and aaiC virulence traits in 27,094 non-diarrheal surveillance stools and 7,692 diarrheal stools from 2,092 children in the MAL-ED birth cohort. We identified risk factors for EAEC and estimated the associations of EAEC with diarrhea, enteropathy biomarker concentrations, and both short-term (one to three months) and long-term growth (to two years of age).

Results

Overall, 9,581 samples (27.5%) were positive for EAEC, and almost all children had at least one detection (94.8%) by two years of age. Exclusive breastfeeding, higher enrollment weight, and macrolide use within the preceding 15 days were protective. Although not associated with diarrhea, EAEC infections were weakly associated with biomarkers of intestinal inflammation and more strongly with reduced length at two years of age (LAZ difference associated with high frequency of EAEC detections: -0.30, 95% CI: -0.44, -0.16).

Conclusions

Asymptomatic EAEC infections were common early in life and were associated with linear growth shortfalls. Associations with intestinal inflammation were small in magnitude, but suggest a pathway for the growth impact. Increasing the duration of exclusive breastfeeding may help prevent these potentially inflammatory infections and reduce the long-term impact of early exposure to EAEC.

Epidemiology, clinical features and risk factors for human rabies and animal bites during an outbreak of rabies in Maputo and Matola cities, Mozambique, 2014: Implications for public health interventions for rabies control

24 July 2017 - 9:00pm

by Cristolde Salomão, Amílcar Nacima, Lutero Cuamba, Lorna Gujral, Olga Amiel, Cynthia Baltazar, Julie Cliff, Eduardo Samo Gudo

Background

In Mozambique, the majority of rabies outbreaks are unreported and data on the epidemiological features of human rabies and animal bites are scarce. An outbreak of human rabies in adjacent Maputo and Matola cities in 2014 prompted us to investigate the epidemiology, clinical features and risk factors of human rabies and animal bites in the two cities.

Methodology/Principal findings

We reviewed cases of human rabies and animal bites from April to July 2014, and carried out a community investigation in July and August in the neighborhoods where cases of human rabies resided. This investigation included collection of clinical, demographic and epidemiological information and a case control study to investigate the risk factors associated with human rabies. Fourteen cases of human rabies were detected in Maputo (n = 10) and Matola (n = 3) cities and neighbouring Boane district (n = 1) between April and August 2014, all of whom had been admitted to hospital. All had a recent history of dog bite. Of the 14 rabid dogs, only one had been immunized. 819 cases of animal bites were registered, of which 64.6% (529/819) were from Maputo City. Dogs were responsible for 97.8% (801/819) of all animal bites, but only 27.0% (126/467) were immunized. Factors significantly associated with human rabies were: age <15 years (p = 0.05), bite by stray dog (p = 0.002), deep wound (p = 0.02), bite in the head (p = 0.001), bite by unimmunized dog (p = 0.01), no use of soap and water (p = 0.001), and no post-exposure prophylaxis (p = 0.01).

Conclusions/Significance

Implementation of control measures for rabies is poor in Maputo and Matola cities, where cases of human rabies were strongly associated with bites by stray and unvaccinated dogs and irregular implementation of post-exposure measures.

A putative ATP/GTP binding protein affects <i>Leishmania mexicana</i> growth in insect vectors and vertebrate hosts

24 July 2017 - 9:00pm

by Aygul Ishemgulova, Natalya Kraeva, Jana Hlaváčová, Sara L. Zimmer, Anzhelika Butenko, Lucie Podešvová, Tereza Leštinová, Julius Lukeš, Alexei Kostygov, Jan Votýpka, Petr Volf, Vyacheslav Yurchenko

Background

Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania’s ability to establish mammalian intracellular infection and to colonize the gut of an insect vector.

Methodology/Principal findings

The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed.

Conclusions/Significance

L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana’s general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.

Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

24 July 2017 - 9:00pm

by Ulrich F. Prodjinotho, Charlotte von Horn, Alex Y. Debrah, Linda Batsa Debrah, Anna Albers, Laura E. Layland, Achim Hoerauf, Tomabu Adjobimey

Helminth parasites are known to be efficient modulators of their host’s immune system. To guarantee their own survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterized. Here we investigate the impact of plasma and affinity-purified IgG/IgG4 fractions from endemic normals (EN) and LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states were investigated by monitoring the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) respectively by flow cytometry and ELISA. We could show that the activation of granulocytes was inhibited in the presence of plasma from EN and Mf+ individuals whereas those of Mf- and CP presented no effect. This inhibitory capacity was impaired upon depletion of IgG in Mf+ individuals but persisted in IgG-depleted plasma from EN, where it strongly correlated with the expression of IgA. In addition, IgA-depleted fractions failed to suppress granulocyte activation. Strikingly, affinity-purified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. Reduction of both the affinity to, and the suppressive properties of anti-inflammatory IgG4 on granulocytes was reached only when FcγRI and II were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP, natively exhibit FcγRI/II-dependent suppressive properties on granulocytes. Our findings suggest that quantitative and qualitative alterations in IgG4 molecules are associated with the different clinical phenotypes in LF endemic regions.

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