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The detection of cryptic <i>Plasmodium</i> infection among villagers in Attapeu province, Lao PDR

20 December 2017 - 10:00pm

by Moritoshi Iwagami, Sengdeuane Keomalaphet, Phonepadith Khattignavong, Pheovaly Soundala, Lavy Lorphachan, Emilie Matsumoto-Takahashi, Michel Strobel, Daniel Reinharz, Manisack Phommasansack, Bouasy Hongvanthong, Paul T. Brey, Shigeyuki Kano

Background

Although the malaria burden in the Lao PDR has gradually decreased, the elimination of malaria by 2030 presents many challenges. Microscopy and malaria rapid diagnostic tests (RDTs) are used to diagnose malaria in the Lao PDR; however, some studies have reported the prevalence of sub-microscopic Plasmodium infections or asymptomatic Plasmodium carriers in endemic areas. Thus, highly sensitive detection methods are needed to understand the precise malaria situation in these areas.

Methodology/Principal findings

A cross-sectional malaria field survey was conducted in 3 highly endemic malaria districts (Xaysetha, Sanamxay, Phouvong) in Attapeu province, Lao PDR in 2015, to investigate the precise malaria endemicity in the area; 719 volunteers from these villages participated in the survey. Microscopy, RDTs and a real-time nested PCR were used to detect Plasmodium infections and their results were compared. A questionnaire survey of all participants was also conducted to estimate risk factors of Plasmodium infection. Numbers of infections detected by the three methods were microscopy: P. falciparum (n = 1), P. vivax (n = 2); RDTs: P. falciparum (n = 2), P. vivax (n = 3); PCR: Plasmodium (n = 47; P. falciparum [n = 4], P. vivax [n = 41], mixed infection [n = 2]; 6.5%, 47/719). Using PCR as a reference, the sensitivity and specificity of microscopy were 33.3% and 100.0%, respectively, for detecting P. falciparum infection, and 7.0% and 100.0%, for detecting P. vivax infection. Among the 47 participants with parasitemia, only one had a fever (≥37.5°C) and 31 (66.0%) were adult males. Risk factors of Plasmodium infection were males and soldiers, whereas a risk factor of asymptomatic Plasmodium infection was a history of ≥3 malaria episodes.

Conclusions/Significance

There were many asymptomatic Plasmodium carriers in the study areas of Attapeu province in 2015. Adult males, probably soldiers, were at high risk for malaria infection. P. vivax, the dominant species, accounted for 87.2% of the Plasmodium infections among the participants. To achieve malaria elimination in the Lao PDR, highly sensitive diagnostic tests, including PCR-based diagnostic methods should be used, and plans targeting high-risk populations and elimination of P. vivax should be designed and implemented.

SYN023, a novel humanized monoclonal antibody cocktail, for post-exposure prophylaxis of rabies

20 December 2017 - 10:00pm

by Tzu-Yuan Chao, Shiqi Ren, Enyun Shen, Susan Moore, Shou-feng Zhang, Li Chen, Charles E. Rupprecht, Eric Tsao

Rabies is a neglected zoonotic disease that is preventable in humans by appropriate post-exposure prophylaxis (PEP). However, current PEP relies on polyclonal immune globulin products purified from pooled human (HRIG) or equine (ERIG) plasma that are either in chronic shortage or in association with safety concerns. Here, we present the development of an antibody cocktail, SYN023, made of two novel monoclonal antibodies (MAb) CTB011 and CTB012 that could serve as safer and more cost-effective alternatives to the current RIG products. Both CTB011 and CTB012 are humanized MAbs that bind to non-overlapping epitopes on the rabies virus (RABV) glycoprotein (G) with sub-nanomolar affinities. Sequence analysis revealed that many of the critical residues in binding are highly conserved across different species of lyssaviruses. When combined at a 1:1 ratio, CTB011/CTB012 exhibited neutralization capabilities equivalent or superior to HRIG against 10 North American street RABV isolates in vitro and 15 prevalent Chinese RABV strains in animal models. Finally, SYN023, at a dosage of 0.03 mg/kg, was able to offer the same degree of protection as standard HRIG administration (20 IU/kg) in Syrian hamsters challenged with a highly virulent bat (Tadarida brasiliensis) RABV variant. Taken together, the high-potency and broad-spectrum neutralization demonstrated by SYN023 make it an effective candidate for human rabies PEP consideration.

Assessment of MALDI-TOF mass spectrometry for filariae detection in <i>Aedes aegypti</i> mosquitoes

20 December 2017 - 10:00pm

by Djamel Tahir, Lionel Almeras, Marie Varloud, Didier Raoult, Bernard Davoust, Philippe Parola

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is an emerging tool for routine identification of bacteria, archaea and fungi. It has also been recently applied as an accurate approach for arthropod identification. Preliminary studies have shown that the MALDI-TOF MS was able to differentiate whether ticks and mosquitoes were infected or not with some bacteria and Plasmodium parasites, respectively. The aim of the present study was to test the efficiency of MALDI-TOF MS tool in distinguishing protein profiles between uninfected mosquitoes from specimens infected by filarioid helminths. Aedes aegypti mosquitoes were engorged on microfilaremic blood infected with Dirofilaria immitis, Brugia malayi or Brugia pahangi. Fifteen days post-infective blood feeding, a total of 534 mosquitoes were killed by freezing. To assess mass spectra (MS) profile changes following filariae infections, one compartment (legs, thorax, head or thorax and head) per mosquito was submitted for MALDI-TOF MS analysis; the remaining body parts were used to establish filariae infectious status by real-time qPCR. A database of reference MS, based on the mass profiles of at least two individual mosquitoes per compartment, was created. Subsequently, the remaining compartment spectra (N = 350) from Ae. aegypti infected or not infected by filariae were blind tested against the spectral database. In total, 37 discriminating peak masses ranging from 2062 to 14869 daltons were identified, of which 17, 11, 12 and 7 peak masses were for legs, thorax, thorax-head and head respectively. Two peak masses (4073 and 8847 Da) were specific to spectra from Ae. aegypti infected with filariae, regardless of nematode species or mosquito compartment. The thorax-head part provided better classification with a specificity of 94.1% and sensitivity of 86.6, 71.4 and 68.7% of D. immitis, B. malayi and B. pahangi respectively. This study presents the potential of MALDI-TOF MS as a reliable tool for differentiating non-infected and filariae-infected Ae. aegypti mosquitoes. Considering that the results might vary in other mosquito species, further studies are needed to consolidate the obtained preliminary results before applying this tool in entomological surveillance as a fast mass screening method of filariosis vectors in endemic areas.

Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the <i>Aedes aegypti</i> midgut

19 December 2017 - 10:00pm

by Vincent Raquin, Sarah Hélène Merkling, Valérie Gausson, Isabelle Moltini-Conclois, Lionel Frangeul, Hugo Varet, Marie-Agnès Dillies, Maria-Carla Saleh, Louis Lambrechts

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

Long-lived CD8+ T cell responses following Crimean-Congo haemorrhagic fever virus infection

19 December 2017 - 10:00pm

by Dominique Goedhals, Janusz T. Paweska, Felicity J. Burt

Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Orthonairovirus genus of the Nairoviridae family and is associated with haemorrhagic fever in humans. Although T lymphocyte responses are known to play a role in protection from and clearance of viral infections, specific T cell epitopes have yet to be identified for CCHFV following infection. A panel of overlapping peptides covering the CCHFV nucleoprotein and the structural glycoproteins, GN and GC, were screened by ELISpot assay to detect interferon gamma (IFN-γ) production in vitro by peripheral blood mononuclear cells from eleven survivors with previous laboratory confirmed CCHFV infection. Reactive peptides were located predominantly on the nucleoprotein, with only one survivor reacting to two peptides from the glycoprotein GC. No single epitope was immunodominant, however all but one survivor showed reactivity to at least one T cell epitope. The responses were present at high frequency and detectable several years after the acute infection despite the absence of continued antigenic stimulation. T cell depletion studies confirmed that IFN-γ production as detected using the ELISpot assay was mediated chiefly by CD8+ T cells. This is the first description of CD8+ T cell epitopic regions for CCHFV and provides confirmation of long-lived T cell responses in survivors of CCHFV infection.

Cost-effectiveness analysis of diarrhoea management approaches in Nigeria: A decision analytical model

19 December 2017 - 10:00pm

by Charles E. Okafor, Obinna I. Ekwunife

Background

Diarrhoea is a leading cause of death in Nigerian children under 5 years. Implementing the most cost-effective approach to diarrhoea management in Nigeria will help optimize health care resources allocation. This study evaluated the cost-effectiveness of various approaches to diarrhoea management namely: the ‘no treatment’ approach (NT); the preventive approach with rotavirus vaccine; the integrated management of childhood illness for diarrhoea approach (IMCI); and rotavirus vaccine plus integrated management of childhood illness for diarrhoea approach (rotavirus vaccine + IMCI).

Methods

Markov cohort model conducted from the payer’s perspective was used to calculate the cost-effectiveness of the four interventions. The markov model simulated a life cycle of 260 weeks for 33 million children under five years at risk of having diarrhoea (well state). Disability adjusted life years (DALYs) averted was used to quantify clinical outcome. Incremental cost-effectiveness ratio (ICER) served as measure of cost-effectiveness.

Results

Based on cost-effectiveness threshold of $2,177.99 (i.e. representing Nigerian GDP/capita), all the approaches were very cost-effective but rotavirus vaccine approach was dominated. While IMCI has the lowest ICER of $4.6/DALY averted, the addition of rotavirus vaccine was cost-effective with an ICER of $80.1/DALY averted. Rotavirus vaccine alone was less efficient in optimizing health care resource allocation.

Conclusion

Rotavirus vaccine + IMCI approach was the most cost-effective approach to childhood diarrhoea management. Its awareness and practice should be promoted in Nigeria. Addition of rotavirus vaccine should be considered for inclusion in the national programme of immunization. Although our findings suggest that addition of rotavirus vaccine to IMCI for diarrhoea is cost-effective, there may be need for further vaccine demonstration studies or real life studies to establish the cost-effectiveness of the vaccine in Nigeria.

Social-ecological factors and preventive actions decrease the risk of dengue infection at the household-level: Results from a prospective dengue surveillance study in Machala, Ecuador

18 December 2017 - 10:00pm

by Aileen Kenneson, Efraín Beltrán-Ayala, Mercy J. Borbor-Cordova, Mark E. Polhemus, Sadie J. Ryan, Timothy P. Endy, Anna M. Stewart-Ibarra

Background

In Ecuador, dengue virus (DENV) infections transmitted by the Aedes aegypti mosquito are among the greatest public health concerns in urban coastal communities. Community- and household-level vector control is the principal means of controlling disease outbreaks. This study aimed to assess the impact of knowledge, attitudes, and practices (KAPs) and social-ecological factors on the presence or absence of DENV infections in the household.

Methods

In 2014 and 2015, individuals with DENV infections from sentinel clinics in Machala, Ecuador, were invited to participate in the study, as well as members of their household and members of four neighboring households located within 200 meters. We conducted diagnostic testing for DENV on all study participants; we surveyed heads of households (HOHs) regarding demographics, housing conditions and KAPs. We compared KAPs and social-ecological factors between households with (n = 139) versus without (n = 80) DENV infections, using bivariate analyses and multivariate logistic regression models with and without interactions.

Results

Significant risk factors in multivariate models included proximity to abandoned properties, interruptions in piped water, and shaded patios (p<0.05). Significant protective factors included the use of mosquito bed nets, fumigation inside the home, and piped water inside the home (p<0.05). In bivariate analyses (but not multivariate modeling), DENV infections were positively associated with HOHs who were male, employed, and of younger age than households without infections (p<0.05). DENV infections were not associated with knowledge, attitude, or reported barriers to prevention activities.

Discussion

Specific actions that can be considered to decrease the risk of DENV infections in the household include targeting vector control in highly shaded properties, fumigating inside the home, and use of mosquito bed nets. Community-level interventions include cleanup of abandoned properties, daily garbage collection, and reliable piped water inside houses. These findings can inform interventions to reduce the risk of other diseases transmitted by the Ae. aegypti mosquito, such as chikungunya and Zika fever.

New insights into <i>Trypanosoma cruzi</i> evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis

18 December 2017 - 10:00pm

by Juan C. Ramírez, Carolina Torres, María de los A. Curto, Alejandro G. Schijman

Trypanosoma cruzi has been subdivided into seven Discrete Typing Units (DTUs), TcI-TcVI and Tcbat. Two major evolutionary models have been proposed to explain the origin of hybrid lineages, but while it is widely accepted that TcV and TcVI are the result of genetic exchange between TcII and TcIII strains, the origin of TcIII and TcIV is still a matter of debate. T. cruzi satellite DNA (SatDNA), comprised of 195 bp units organized in tandem repeats, from both TcV and TcVI stocks were found to have SatDNA copies type TcI and TcII; whereas contradictory results were observed for TcIII stocks and no TcIV sequence has been analyzed yet. Herein, we have gone deeper into this matter analyzing 335 distinct SatDNA sequences from 19 T. cruzi stocks representative of DTUs TcI-TcVI for phylogenetic inference. Bayesian phylogenetic tree showed that all sequences were grouped in three major clusters, which corresponded to sequences from DTUs TcI/III, TcII and TcIV; whereas TcV and TcVI stocks had two sets of sequences distributed into TcI/III and TcII clusters. As expected, the lowest genetic distances were found between TcI and TcIII, and between TcV and TcVI sequences; whereas the highest ones were observed between TcII and TcI/III, and among TcIV sequences and those from the remaining DTUs. In addition, signature patterns associated to specific T. cruzi lineages were identified and new primers that improved SatDNA-based qPCR sensitivity were designed. Our findings support the theory that TcIII is not the result of a hybridization event between TcI and TcII, and that TcIV had an independent origin from the other DTUs, contributing to clarifying the evolutionary history of T. cruzi lineages. Moreover, this work opens the possibility of typing samples from Chagas disease patients with low parasitic loads and improving molecular diagnostic methods of T. cruzi infection based on SatDNA sequence amplification.

Human Neutrophil Peptide 1 as immunotherapeutic agent against <i>Leishmania</i> infected BALB/c mice

18 December 2017 - 10:00pm

by Zahra Abdossamadi, Negar Seyed, Farnaz Zahedifard, Tahereh Taheri, Yasaman Taslimi, Hossein Montakhab-Yeganeh, Alireza Badirzadeh, Mohammad Vasei, Safoora Gharibzadeh, Sima Rafati

Human Neutrophil Peptide-1 (HNP1) produced by neutrophils, is a well-known antimicrobial peptide which plays a role both in innate as well as in adaptive immunity and is under intensive investigation as a potential therapeutic agent. Previous in vitro experiments have indicated the leishmaniacidal effect of recombinant HNP1 on Leishmania major (L. major) promastigotes and amastigotes. In the current study, we further extended the idea to explore the remedial effect of HNP1 in the two modalities of peptide therapy (folded HNP1) and gene therapy in L. major infected BALB/c mice. To this end, mice in five different groups received synthetic folded HNP1 (G1), pcDNA-HNP1-EGFP (G2), pcDNA-EGFP (G3), Amphotericin B (G4) and PBS (G5), which was started three weeks after infection for three consecutive weeks. Footpad swelling was monitored weekly and a day after the therapy ended, IFN-γ, IL-4, IL-10, IL-6 and nitric oxide produced by splenocytes were analyzed together with the parasite load in draining lymph nodes. Arginase activity and dermal histopathological changes were also analyzed in the infected footpads. We demonstrated that both therapeutic approaches effectively induced Th1 polarization and restricted parasite burden. It can control disease progression in contrast to non-treated groups. However, pcDNA-HNP1-EGFP is more promising in respect to parasite control than folded HNP1, but less effective than AmB treatment. We concluded with the call for a future approach, that is, a DNA-based expression of HNP1 combined with AmB as it can improve the leishmaniacidal efficacy.

Host immunity, nutrition and coinfection alter longitudinal infection patterns of schistosomes in a free ranging African buffalo population

18 December 2017 - 10:00pm

by Brianna R. Beechler, Anna E. Jolles, Sarah A. Budischak, Paul L. A. M. Corstjens, Vanessa O. Ezenwa, Mireya Smith, Robert S. Spaan, Govert J. van Dam, Michelle L. Steinauer

Schistosomes are trematode parasites of global importance, causing infections in millions of people, livestock, and wildlife. Most studies on schistosomiasis, involve human subjects; as such, there is a paucity of longitudinal studies investigating parasite dynamics in the absence of intervention. As a consequence, despite decades of research on schistosomiasis, our understanding of its ecology in natural host populations is centered around how environmental exposure and acquired immunity influence acquisition of parasites, while very little is known about the influence of host physiology, coinfection and clearance in the absence of drug treatment. We used a 4-year study in free-ranging African buffalo to investigate natural schistosome dynamics. We asked (i) what are the spatial and temporal patterns of schistosome infections; (ii) how do parasite burdens vary over time within individual hosts; and (iii) what host factors (immunological, physiological, co-infection) and environmental factors (season, location) explain patterns of schistosome acquisition and loss in buffalo? Schistosome infections were common among buffalo. Microgeographic structure explained some variation in parasite burdens among hosts, indicating transmission hotspots. Overall, parasite burdens ratcheted up over time; however, gains in schistosome abundance in the dry season were partially offset by losses in the wet season, with some hosts demonstrating complete clearance of infection. Variation among buffalo in schistosome loss was associated with immunologic and nutritional factors, as well as co-infection by the gastrointestinal helminth Cooperia fuelleborni. Our results demonstrate that schistosome infections are surprisingly dynamic in a free-living mammalian host population, and point to a role for host factors in driving variation in parasite clearance, but not parasite acquisition which is driven by seasonal changes and spatial habitat utilization. Our study illustrates the power of longitudinal studies for discovering mechanisms underlying parasite dynamics in individual animals and populations.

Increased activated memory B-cells in the peripheral blood of patients with erythema nodosum leprosum reactions

18 December 2017 - 10:00pm

by Edessa Negera, Stephen L. Walker, Yonas Bekele, Hazel M. Dockrell, Diana N. Lockwood

B-cells, in addition to antibody secretion, have emerged increasingly as effector and immunoregulatory cells in several chronic inflammatory diseases. Although Erythema Nodosum Leprosum (ENL) is an inflammatory complication of leprosy, the role of B- cell subsets has never been studied in this patient group. Therefore, it would be interesting to examine the contribution of B-cells in the pathogenesis of ENL. A case-control study design was used to recruit 30 untreated patients with ENL and 30 non-reactional lepromatous leprosy (LL) patient controls at ALERT Hospital, Ethiopia. Peripheral blood samples were obtained before, during and after treatment from each patient. Peripheral blood mononuclear cells (PBMCs) were isolated and used for immunophenotyping of B- cell subsets by flow cytometry. The kinetics of B-cells in patients with ENL before, during and after Prednisolone treatment of ENL was compared with LL patient controls as well as within ENL group. Total B-cells, mature B-cells and resting memory B-cells were not significantly different between patients with ENL reactions and LL controls before treatment. Interestingly, while the percentage of naive B-cells was significantly lower in untreated ENL patients than in LL patient controls, the percentage of activated memory B-cells was significantly higher in these untreated ENL patients than in LL controls. On the other hand, the percentage of tissue-like memory B-cells was considerably low in untreated ENL patients compared to LL controls. It appears that the lower frequency of tissue-like memory B-cells in untreated ENL could promote the B-cell/T-cell interaction in these patients through downregulation of inhibitory molecules unlike in LL patients. Conversely, the increased production of activated memory B-cells in ENL patients could imply the scale up of immune activation through antigen presentation to T-cells. However, the generation and differential function of these memory B-cells need further investigation. The finding of increased percentage of activated memory B-cells in untreated patients with ENL reactions suggests the association of these cells with the ENL pathology. The mechanism by which inflammatory reactions like ENL affecting these memory cells and contributing to the disease pathology is an interesting area to be explored for and could lead to the development of novel and highly efficacious drug for ENL treatment.

Analysis of the efficacy of Taiwanese freeze-dried neurotoxic antivenom against <i>Naja kaouthia</i>, <i>Naja siamensis</i> and <i>Ophiophagus hannah</i> through proteomics and animal model approaches

15 December 2017 - 10:00pm

by Chien-Chun Liu, Chen-Hsien You, Po-Jung Wang, Jau-Song Yu, Guo-Jen Huang, Chien-Hsin Liu, Wen-Chin Hsieh, Chih-Chuan Lin

In Southeast Asia, envenoming resulting from cobra snakebites is an important public health issue in many regions, and antivenom therapy is the standard treatment for the snakebite. Because these cobras share a close evolutionary history, the amino acid sequences of major venom components in different snakes are very similar. Therefore, either monovalent or polyvalent antivenoms may offer paraspecific protection against envenomation of humans by several different snakes. In Taiwan, a bivalent antivenom—freeze-dried neurotoxic antivenom (FNAV)—against Bungarus multicinctus and Naja atra is available. However, whether this antivenom is also capable of neutralizing the venom of other species of snakes is not known. Here, to expand the clinical application of Taiwanese FNAV, we used an animal model to evaluate the neutralizing ability of FNAV against the venoms of three common snakes in Southeast Asia, including two ‘true’ cobras Naja kaouthia (Thailand) and Naja siamensis (Thailand), and the king cobra Ophiophagus hannah (Indonesia). We further applied mass spectrometry (MS)-based proteomic techniques to characterize venom proteomes and identify FNAV-recognizable antigens in the venoms of these Asian snakes. Neutralization assays in a mouse model showed that FNAV effectively neutralized the lethality of N. kaouthia and N. siamensis venoms, but not O. hannah venom. MS-based venom protein identification results further revealed that FNAV strongly recognized three-finger toxin and phospholipase A2, the major protein components of N. kaouthia and N. siamensis venoms. The characterization of venom proteomes and identification of FNAV-recognizable venom antigens may help researchers to further develop more effective antivenom designed to block the toxicity of dominant toxic proteins, with the ultimate goal of achieving broadly therapeutic effects against these cobra snakebites.

Genotyping of <i>Mycobacterium leprae</i> for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil

15 December 2017 - 10:00pm

by Amanda N. B. Fontes, Luana N. G. C. Lima, Rosa M. S. Mota, Rosa L. F. Almeida, Maria A. Pontes, Heitor de S. Gonçalves, Cristiane C. Frota, Varalakshmi D. Vissa, Patrick J. Brennan, Ricardo J. P. S. Guimaraes, Carl Kendall, Ligia R. F. S. Kerr, Philip N. Suffys

Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1–4 typing of Mycobacterium leprae in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from M. leprae in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique M. leprae genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation.

Polypyridylruthenium(II) complexes exert anti-schistosome activity and inhibit parasite acetylcholinesterases

14 December 2017 - 10:00pm

by Madhu K. Sundaraneedi, Bemnet Tedla, Ramon M. Eichenberger, Luke Becker, Darren Pickering, Michael J. Smout, Siji Rajan, Phurpa Wangchuk, F. Richard Keene, Alex Loukas, J. Grant Collins, Mark S. Pearson

Background

Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)—the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs.

Methodology/Principal findings

We investigated the anti-schistosome efficacy of polypyridylruthenium(II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb12-tri and Rubb7-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb12-tri) and 10 mg/kg (Rubb7-tnl). Mice treated with Rubb12-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb12-tri—68%, Rubb7-tnl—56%) and were significantly morphologically altered (Rubb12-tri—62% abnormal, Rubb7-tnl—35% abnormal). We hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway.

Conclusions/Significance

Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes.

Virulence, pathology, and pathogenesis of Pteropine orthoreovirus (PRV) in BALB/c mice: Development of an animal infection model for PRV

14 December 2017 - 10:00pm

by Kazutaka Egawa, Masayuki Shimojima, Satoshi Taniguchi, Noriyo Nagata, Hideki Tani, Tomoki Yoshikawa, Takeshi Kurosu, Shumpei Watanabe, Shuetsu Fukushi, Masayuki Saijo

Background

Cases of acute respiratory tract infection caused by Pteropine orthoreovirus (PRV) of the genus Orthoreovirus (family: Reoviridae) have been reported in Southeast Asia, where it was isolated from humans and bats. It is possible that PRV-associated respiratory infections might be prevalent in Southeast Asia. The clinical course of PRV is not fully elucidated.

Methods

The virulence, pathology, and pathogenesis of two PRV strains, a human-borne PRV strain (isolated from a patient, who returned to Japan from Bali, Indonesia in 2007) and a bat-borne PRV (isolated from a bat [Eonycteris spelaea] in the Philippines in 2013) were investigated in BALB/c mice using virological, pathological, and immunological study methods.

Results

The intranasal inoculation of BALB/c mice with human-borne PRV caused respiratory infection. In addition, all mice with immunity induced by pre-inoculation with a non-lethal dose of PRV were completely protected against lethal PRV infection. Mice treated with antiserum with neutralizing antibody activity after inoculation with a lethal dose of PRV showed a reduced fatality rate. In this mouse model, bat-borne PRV caused respiratory infection similar to human-borne PRV. PRV caused lethal respiratory disease in an animal model of PRV infection, in which BALB/c mice were used.

Conclusions

The BALB/c mouse model might help to accelerate research on the virulence of PRV and be useful for evaluating the efficacy of therapeutic agents and vaccines for the treatment and prevention of PRV infection. PRV was shown for the first time to be a causative virus of respiratory disease on the basis of Koch’s postulations by the additional demonstration that PRV caused respiratory disease in mice through their intranasal inoculation with PRV.

Drug resistance and treatment failure in leishmaniasis: A 21st century challenge

14 December 2017 - 10:00pm

by Alicia Ponte-Sucre, Francisco Gamarro, Jean-Claude Dujardin, Michael P. Barrett, Rogelio López-Vélez, Raquel García-Hernández, Andrew W. Pountain, Roy Mwenechanya, Barbara Papadopoulou

Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of “resistance” related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.

Emergence of melioidosis in the Indian Ocean region: Two new cases and a literature review

14 December 2017 - 10:00pm

by Nicolas Allou, Olivier Martinet, Jérôme Allyn, Bruno Bouchet, Marie-Christine Jaffar-Bandjee, Thomas Galas, Nicolas Traversier, Olivier Belmonte

Author summary: Melioidosis is a disease caused by bacteria called B. pseudomallei. Infections can develop after contact with standing water. This disease can reach all the organs and especially the lungs. It is associated with a high mortality rate (up to 50%). Melioidosis is endemic in northern Australia and in Southeast Asia. Nevertheless, B. pseudomallei may be endemic in the Indian Ocean region and in Madagascar in particular, so clinicians and microbiologists should consider acute melioidosis as a differential diagnosis in the Indian Ocean region, in particular from Madagascar.

RNA interference in <i>Fasciola gigantica</i>: Establishing and optimization of experimental RNAi in the newly excysted juveniles of the fluke

12 December 2017 - 10:00pm

by Arun Anandanarayanan, Opinder Krishen Raina, Hniang Lalrinkima, Ajayta Rialch, Muthu Sankar, Anju Varghese

Fasciolosis caused by Fasciola gigantica is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa. Resistance to commonly used anthelmintics in Fasciola has increased the need to search for alternative therapeutic targets. RNA interference is the current tool of choice in the search for such targets in Fasciola. The susceptibility of juvenile Fasciola hepatica to double stranded (ds) RNA induced RNAi has been established but in F. gigantica a single preliminary report on RNAi induced mRNA transcript knockdown is available. Here we optimized conditions for RNAi in the liver fluke F.gigantica targeting six genes including superoxide dismutase (SOD), σ class of glutathione-s-transferase (GST), cathepsin (Cat) L1-D, Cat B1, Cat B2 and Cat B3 that showed robust transcriptional silencing of the targets following exposure of the newly excysted juveniles (NEJs) to long (170–223 nt) dsRNA. Knockdown was shown to be concentration dependent with significant mRNA transcript suppression occurring at 5 ng / μl that showed further suppression with the increase in the dsRNA concentration. The dsRNA induced persistent silencing of the mRNA transcript of SOD and σGST up to 15 days of observation. Delivery of the long dsRNA and siRNA to the newly excysted juveniles by soaking method was found to be efficient by tracking the uptake and diffusion of Cy3 labelled siRNA and long dsRNA in the flukes. Off-target effects of dsRNA trigger on some of the non-target genes were detected in the present investigation on RNAi in F. gigantica. The dsRNA induced superoxide dismutase protein suppression while impact of RNAi on other target proteins was not studied. There is no in vitro culture system for prolonged survival of the F. gigantica and in the present study in vitro maintenance of the NEJs is reported for a period of 3 weeks. The present study is the first attempt on optimization of RNAi protocols in F. gigantica where long dsRNA allowed for an efficient and persistent gene silencing, opening prospects for functional validation of putative vaccine and therapeutic targets in this neglected parasite.

Elimination of trachoma as a public health problem in Ghana: Providing evidence through a pre-validation survey

12 December 2017 - 10:00pm

by Oscar Debrah, Ernest O. Mensah, Laura Senyonjo, Dziedzom K. de Souza, Tei E. Hervie, David Agyemang, Didier Bakajika, Benjamin Marfo, Felix Ahorsu, Seth Wanye, Robin Bailey, Joseph B. Koroma, Agatha Aboe, Nana-Kwadwo Biritwum

Background

In order to achieve elimination of trachoma, a country needs to demonstrate that the elimination prevalence thresholds have been achieved and then sustained for at least a two-year period. Ghana achieved the thresholds in 2008, and since 2011 has been implementing its trachoma surveillance strategy, which includes community and school screening for signs of follicular trachoma and trichiasis, in trachoma-endemic districts. In 2015–2016, the country conducted a district level population-based survey to validate elimination of trachoma as a public health problem.

Methods

As per WHO recommendations, a cross-sectional survey, employing a two-stage cluster random sampling methodology, was used across 18 previously trachoma endemic districts (evaluation units (EUs) in the Upper West and Northern Regions of Ghana. In each EU 24 villages were selected based on probability proportional to estimated size. A minimum of 40 households were targeted per village and all eligible residents were examined for clinical signs of trachoma, using the WHO simplified grading system. The number of trichiasis cases unknown to the health system was determined. Household environmental risk factors for trachoma were also assessed.

Results

Data from 45,660 individuals were examined from 11,099 households across 18 EUs, with 27,398 (60.0%) children aged 1–9 years and 16,610 (36.4%) individuals 15 years and above All EUs had shown to have maintained the WHO elimination threshold for Trachomatous inflammation-Follicular (TF) (<5.0% prevalence) in children aged 1–9 years old. The EU TF prevalence in children aged 1–9 years old ranged from between 0.09% to 1.20%. Only one EU (Yendi 0.36%; 95% CI: 0.0–1.01) failed to meet the WHO TT elimination threshold (< 0.2% prevalence in adults aged 15 and above). The EU prevalence of trichiasis (TT) unknown to the health system in adults aged ≥15 years, ranged from 0.00% to 0.36%. In this EU, the estimated TT backlog is 417 All TT patients identified in the study, as well as through on-going surveillance efforts will require further management. A total of 75.9% (95% CI 72.1–79.3, EU range 29.1–92.6) of households defecated in the open but many households had access to an improved water source 75.9% (95%CI: 71.5–79.8, EU range 47.4–90.1%), with 45.5% (95% CI 41.5–49.7%, EU range 28.4–61.8%) making a round trip of water collection < 30 minutes.

Conclusion

The findings from this survey indicate elimination thresholds have been maintained in Ghana in 17 of the 18 surveyed EUs. Only one EU, Yendi, did not achieve the TT elimination threshold. A scheduled house-by-house TT case search in this EU coupled with surgery to clear the backlog of cases is necessary in order for Ghana to request validation of elimination of trachoma as a public health problem.

Trachomatous scarring among children in a formerly hyper-endemic district of Tanzania

12 December 2017 - 10:00pm

by Jacob T. Cox, Harran Mkocha, Beatriz Munoz, Sheila K. West

Background

Associations between repeated ocular infections with Chlamydia trachomatis in childhood and conjunctival scarring in adulthood are well established. Trachomatous scarring (TS) in children has also been observed in hyper-endemic areas, but data are scant regarding childhood scarring in areas where trachoma has been reduced to hypo-endemic levels.

Methods/Principle findings

In this cross-sectional study, a random sample of children, ages 1–9 years, were selected from 38 communities in the formerly hyper-endemic district of Kongwa, Tanzania. Each participant received an ocular examination and eye-swab test for C. trachomatis infection. Conjunctival photographs were taken and analyzed at 5x magnification to determine scarring presence and severity. Community-level case clustering was assessed using intra-class correlation coefficients, and associations between TS presence and demographic/clinical factors were assessed using contingency table analyses. 1,496 children (78% of eligible) participated in this study. The mean age was 5.5 years and 51% were female. Scarring prevalence was 2.1% (95% CI: 1.5%– 3.0%). The prevalence of follicular trachoma and ocular C. trachomatis infection were 3.2% and 6.5%, respectively. Most TS cases (68.7%) fell into the mildest category, grade S1. 18.7% were grade S2; 12.6% were grade S3. No significant associations were seen between TS presence and age, sex, follicular trachoma, or active ocular C. trachomatis infection (p-values: 0.14, 0.48, 0.27, 0.15, respectively). Thirty communities (78.9%) had 0–1 TS cases, and the most seen in any single community was four cases. Three years ago, follicular trachoma prevalence averaged 4.9% in communities with 0–1 TS cases, but 7.6% in communities with 2–4 TS cases (p-value: 0.08).

Conclusions

In this formerly hyper-endemic district of Tanzania, TS was rare in 1–9 year-olds and usually mild when present. Communities with higher rates of follicular trachoma in the past were more likely to have ≥2 cases of scarring, but the association was not statistically significant.

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