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Malaria vector species in Amazonian Peru co-occur in larval habitats but have distinct larval microbial communities

PLoS Neglected Tropical Diseases News - 15 May 2019 - 9:00pm

by Catharine Prussing, Marlon P. Saavedra, Sara A. Bickersmith, Freddy Alava, Mitchel Guzmán, Edgar Manrique, Gabriel Carrasco-Escobar, Marta Moreno, Dionicia Gamboa, Joseph M. Vinetz, Jan E. Conn

In Amazonian Peru, the primary malaria vector, Nyssorhynchus darlingi (formerly Anopheles darlingi), is difficult to target using standard vector control methods because it mainly feeds and rests outdoors. Larval source management could be a useful supplementary intervention, but to determine its feasibility, more detailed studies on the larval ecology of Ny. darlingi are essential. We conducted a multi-level study of the larval ecology of Anophelinae mosquitoes in the peri-Iquitos region of Amazonian Peru, examining the environmental characteristics of the larval habitats of four species, comparing the larval microbiota among species and habitats, and placing Ny. darlingi larval habitats in the context of spatial heterogeneity in human malaria transmission. We collected Ny. darlingi, Nyssorhynchus rangeli (formerly Anopheles rangeli), Nyssorhynchus triannulatus s.l. (formerly Anopheles triannulatus s.l.), and Nyssorhynchus sp. nr. konderi (formerly Anopheles sp. nr. konderi) from natural and artificial water bodies throughout the rainy and dry seasons. We found that, consistent with previous studies in this region and in Brazil, the presence of Ny. darlingi was significantly associated with water bodies in landscapes with more recent deforestation and lower light intensity. Nyssorhynchus darlingi presence was also significantly associated with a lower vegetation index, other Anophelinae species, and emergent vegetation. Though they were collected in the same water bodies, the microbial communities of Ny. darlingi larvae were distinct from those of Ny. rangeli and Ny. triannulatus s.l., providing evidence either for a species-specific larval microbiome or for segregation of these species in distinct microhabitats within each water body. We demonstrated that houses with more reported malaria cases were located closer to Ny. darlingi larval habitats; thus, targeted control of these sites could help ameliorate malaria risk. The co-occurrence of Ny. darlingi larvae in water bodies with other putative malaria vectors increases the potential impact of larval source management in this region.

Interplay between proinflammatory cytokines, miRNA, and tissue lesions in <i>Anisakis</i>-infected Sprague-Dawley rats

PLoS Neglected Tropical Diseases News - 15 May 2019 - 9:00pm

by Jerko Hrabar, Željka Trumbić, Ivana Bočina, Ivana Bušelić, Anamarija Vrbatović, Ivona Mladineo


Anisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases.

Methodology/Principal findings

The aim of this study was to characterize the early cellular (6–72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM); ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, iNOS+, and S100A8/A9+ cells. The greatest number of iNOS+ and S100A8/A9+ cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls.


Anisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans.

A 15-year review of dengue hospitalizations in Singapore: Reducing admissions without adverse consequences, 2003 to 2017

PLoS Neglected Tropical Diseases News - 15 May 2019 - 9:00pm

by Li Wei Ang, Tun-Linn Thein, Yixiang Ng, Irving Charles Boudville, Po Ying Chia, Vernon Jian Ming Lee, Yee-Sin Leo


Since the 1990s, Singapore has experienced periodic dengue epidemics of increasing frequency and magnitude. In the aftermath of the 2004–2005 dengue epidemic, hospitals refined their admission criteria for dengue cases to right-site dengue case management and reduce the burden of healthcare utilization and negative outcomes. In this study, we describe the national trends of hospital admissions for dengue and disease severity in terms of length of stay (LOS), admission to the intensive care unit (ICU) and death in hospital, and case fatality rate (CFR) in Singapore.


We conducted a retrospective study of notified cases and laboratory confirmed dengue patients admitted to all public and private hospitals between 2003 and 2017. Case notifications for dengue and hospitalization records were extracted from national databases.


The proportion of dengue cases hospitalized was lower in recent years; 28.9% in the 2013–2014 epidemic, compared to 93.2% in the 2004–2005 epidemic, and 58.1% in the 2007 epidemic. Median LOS remained stable over the years; overall LOS was 3 to 4 days and ICU stay was 2 to 3 days. Less than 2% of hospitalized patients were admitted to the ICU. Overall CFR was low and remained below 0.5%. The proportions of dengue cases hospitalized and patients admitted to the ICU were highest in the elderly aged 65 years and older.


While the proportion of dengue cases hospitalized saw a drastic decline due to more selective admission criteria, there was no concomitant increase in adverse outcomes, suggesting that admission criteria were appropriate to focus on severe dengue cases. Further studies are needed to optimize dengue management in older adults who are more likely to be hospitalized with greater disease severity, given the higher proportions of hospitalizations and severe disease among older adults.

In-depth proteomic characterization of <i>Schistosoma haematobium</i>: Towards the development of new tools for elimination

PLoS Neglected Tropical Diseases News - 15 May 2019 - 9:00pm

by Javier Sotillo, Mark S. Pearson, Luke Becker, Gebeyaw G. Mekonnen, Abena S. Amoah, Govert van Dam, Paul L. A. M. Corstjens, Janice Murray, Takafira Mduluza, Francisca Mutapi, Alex Loukas


Schistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogential clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease.

Methods and findings

By combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection.


We provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease.

Implications of environmental and pathogen-specific determinants on clinical presentations and disease outcome in melioidosis patients

PLoS Neglected Tropical Diseases News - 15 May 2019 - 9:00pm

by Tushar Shaw, Chaitanya Tellapragada, Asha Kamath, Vandana Kalwaje Eshwara, Chiranjay Mukhopadhyay


Melioidosis is gaining recognition as an emerging infectious disease with diverse clinical manifestations and high-case fatality rates worldwide. However, the molecular epidemiology of the disease outside the endemic regions such as northeast part of Thailand and northern Australia remains unclear.

Methodology/Principal findings

Clinical data and B. pseudomallei isolates obtained from 199 culture-confirmed cases of melioidosis diagnosed during 2006–2016 in South India were used to elucidate the host and pathogen specific variable virulence determinants associated with clinical presentations and disease outcome. Further, we determined the temporal variations and the influence of ecological factors on B.pseudomallei Lipopolysaccharide (LPS) genotypes causing infections. Severe forms of the disease were observed amongst 169 (85%) patients. Renal dysfunction and infection due to B.pseudomallei harboring BimABm variant had significant associations with severe forms of the disease. Diabetes mellitus, septicemic melioidosis and infection due to LPSB genotype were independent risk factors for mortality. LPSB (74%) and LPSA (20.6%) were the prevalent genotypes causing infections. Both genotypes demonstrated temporal variations and had significant correlations with rainfall and humidity.


Our study findings suggest that the pathogen specific virulence traits under the influence of ecological factors are the key drivers for geographical variations in the molecular epidemiology of melioidosis.

Provision of deworming intervention to pregnant women by antenatal services in countries endemic for soil-transmitted helminthiasis

PLoS Neglected Tropical Diseases News - 13 May 2019 - 9:00pm

by Mathieu Bangert, Pilar Bancalari, Denise Mupfasoni, Alexei Mikhailov, Albis F. Gabrielli, Antonio Montresor


The World Health Organization has recently reemphasized the importance of providing preventive chemotherapy to women of reproductive age in countries endemic for soil-transmitted helminthiasis as they are at heightened risk of associated morbidity. The Demographic and Health Surveys (DHS) Program is responsible for collecting and disseminating accurate, nationally representative data on health and population in developing countries. Our study aims to estimate the number of pregnant women at risk of soil-transmitted helminthiasis that self-reported deworming by antenatal services in endemic countries that conducted Demographic and Health Surveys.

Methodology/Principal findings

The number of pregnant women living in endemic countries was extrapolated from the United Nations World Population Prospects 2015. National deworming coverage among pregnant women were extracted from Demographic and Health Surveys and applied to total numbers of pregnant women in the country.Sub-national DHS with data on self-reported deworming were available from 49 of the 102 endemic countries. In some regions more than 73% of STH endemic countries had a DHS. The DHS report an average deworming coverage of 23% (CI 19–28), ranging from 2% (CI 1–3) to 35% (CI 29–40) in the different regions, meaning more than 16 million pregnant women were dewormed in countries surveyed by DHS. The deworming rates amongst the 43 million pregnant women in STH endemic countries not surveyed by DHS remains unknown.


These estimates will serve to establish baseline numbers of deworming coverage among pregnant women, monitor progress, and urge endemic countries to continue working toward reducing the burden of soil-transmitted helminthiasis. The DHS program should be extended to STH-endemic countries currently not covering the topic of deworming during pregnancy.

Analysis of host cell binding specificity mediated by the Tp0136 adhesin of the syphilis agent <i>Treponema pallidum</i> subsp. <i>pallidum</i>

by Vitomir Djokic, Lorenzo Giacani, Nikhat Parveen


Syphilis affects approximately 11 million people each year globally, and is the third most prevalent sexually transmitted bacterial infection in the United States. Inability to independently culture and genetically manipulate Treponema pallidum subsp. pallidum, the causative agent of this disease, has hindered our understanding of the molecular mechanisms of syphilis pathogenesis. Here, we used the non-infectious and poorly adherent B314 strain of the Lyme disease-causing spirochete, Borrelia burgdorferi, to express two variants of a known fibronectin-binding adhesin, Tp0136, from T. pallidum SS14 and Nichols strains. Using this surrogate system, we investigated the ability of Tp0136 in facilitating differential binding to mammalian cell lines offering insight into the possible role of this virulence factor in colonization of specific tissues by T. pallidum during infection.

Principal findings

Expression of Tp0136 could be detected on the surface of B. burgdorferi by indirect immunofluorescence assay using sera from a secondary syphilis patient that does not react with intact B314 spirochetes transformed with the empty vector. Increase in Tp0136-mediated adherence of B314 strain to human epithelial HEK293 cells was observed with comparable levels of binding exhibited by both Tp0136 alleles. Adherence of Tp0136-expressing B314 was highest to epithelial HEK293 and C6 glioma cells. Gain in binding of B314 strain expressing Tp0136 to purified fibronectin and poor binding of these spirochetes to the fibronectin-deficient cell line (HEp-2) indicated that Tp0136 interaction with this host receptor plays an important role in spirochetal attachment to mammalian cells. Furthermore, preincubation of these cell lines with fibronectin-binding peptide from Staphylococcus aureus FnbA-2 protein significantly inhibited binding of B314 expressing Tp0136.


Our results show that Tp0136 facilitates differential level of binding to cell lines representing various host tissues, which highlights the importance of this protein in colonization of human organs by T. pallidum and resulting syphilis pathogenesis.

Modelling population dynamics and seasonal movement to assess and predict the burden of melioidosis

by Wiriya Mahikul, Lisa J. White, Kittiyod Poovorawan, Ngamphol Soonthornworasiri, Pataporn Sukontamarn, Phetsavanh Chanthavilay, Graham F. Medley, Wirichada Pan-ngum


Melioidosis is an infectious disease that is transmitted mainly through contact with contaminated soil or water, and exhibits marked seasonality in most settings, including Southeast Asia. In this study, we used mathematical modelling to examine the impacts of such demographic changes on melioidosis incidence, and to predict the disease burden in a developing country such as Thailand.

Methodology/Principal findings

A melioidosis infection model was constructed which included demographic data, diabetes mellitus (DM) prevalence, and melioidosis disease processes. The model was fitted to reported melioidosis incidence in Thailand by age, sex, and geographical area, between 2008 and 2015, using a Bayesian Markov Chain Monte Carlo (MCMC) approach. The model was then used to predict the disease burden and future trends of melioidosis incidence in Thailand. Our model predicted two-fold higher incidence rates of melioidosis compared with national surveillance data from 2015. The estimated incidence rates among males were two-fold greater than those in females. Furthermore, the melioidosis incidence rates in the Northeast region population, and among the transient population, were more than double compared to the non-Northeast region population. The highest incidence rates occurred in males aged 45–59 years old for all regions. The average incidence rate of melioidosis between 2005 and 2035 was predicted to be 11.42 to 12.78 per 100,000 population per year, with a slightly increasing trend. Overall, it was estimated that about half of all cases of melioidosis were symptomatic. In addition, the model suggested a greater susceptibility to melioidosis in diabetic compared with non-diabetic individuals.


The increasing trend of melioidosis incidence rates was significantly higher among working-age Northeast and transient populations, males aged ≥45 years old, and diabetic individuals. Targeted intervention strategies, such as health education and awareness raising initiatives, should be implemented on high-risk groups, such as those living in the Northeast region, and the seasonally transient population.

Functional genomics in sand fly–derived <i>Leishmania</i> promastigotes

by Pedro J. Alcolea, Ana Alonso, Ricardo Molina, Maribel Jiménez, Peter J. Myler, Vicente Larraga


Leishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression–profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly–and culture-derived promastigotes.


Transcriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly–derived promastigotes have been found. Sand fly–derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein (HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly–derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut.


Transcriptome analysis has been demonstrated to be technically efficacious to study differential gene expression in sand fly gut promastigote forms. Transcript and protein levels are not well correlated in these organisms (approximately 25% quantitative coincidences), especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view (increase, decrease, or no variation). Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly–derived promastigotes so far.

<i>Cinnamomum cassia</i> exhibits antileishmanial activity against <i>Leishmania donovani</i> infection <i>in vitro</i> and <i>in vivo</i>

by Farhat Afrin, Garima Chouhan, Mohammad Islamuddin, Muzamil Y. Want, Hani A. Ozbak, Hassan A. Hemeg


There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia.


Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS).

Principal findings

CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 μg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 μg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect.


Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial.

Pediatric tropical medicine: The neglected diseases of children

by Peter J. Hotez, Audrey R. Odom John, A. Desiree LaBeaud

Flagellin-independent effects of a Toll-like receptor 5 polymorphism in the inflammatory response to <i>Burkholderia pseudomallei</i>

by Amy K. Dickey, Narisara Chantratita, Sarunporn Tandhavanant, Deirdre Ducken, Lara Lovelace-Macon, Sudeshna Seal, Johanna Robertson, Nicolle D. Myers, Sandra Schwarz, Mark M. Wurfel, Susanna Kosamo, T. Eoin West


Toll-like receptors (TLRs) are sentinel receptors of the innate immune system. TLR4 detects bacterial lipopolysaccharide (LPS) and TLR5 detects bacterial flagellin. A common human nonsense polymorphism, TLR5:c.1174C>T, results in a non-functional TLR5 protein. Individuals carrying this variant have decreased mortality from melioidosis, infection caused by the flagellated Gram-negative bacterium Burkholderia pseudomallei. Although impaired flagellin-dependent signaling in carriers of TLR5:c.1174C>T is well established, this study tested the hypothesis that a functional effect of TLR5:c.1174C>T is flagellin-independent and involves LPS-TLR4 pathways.

Methodology/Principal findings

Whole blood from two independent cohorts of individuals genotyped at TLR5:c.1174C>T was stimulated with wild type or aflagellated B. pseudomallei or purified bacterial motifs followed by plasma cytokine measurements. Blood from individuals carrying the TLR5:c.1174C>T variant produced less IL-6 and IL-10 in response to an aflagellated B. pseudomallei mutant and less IL-8 in response to purified B. pseudomallei LPS than blood from individuals without the variant. TLR5 expression in THP1 cells was silenced using siRNA; these cells were stimulated with LPS before cytokine levels in cell supernatants were quantified by ELISA. In these cells following LPS stimulation, silencing of TLR5 with siRNA reduced both TNF-α and IL-8 levels. These effects were not explained by differences in TLR4 mRNA expression or NF-κB or IRF activation.


The effects of the common nonsense TLR5:c.1174C>T polymorphism on the host inflammatory response to B. pseudomallei may not be restricted to flagellin-driven pathways. Moreover, TLR5 may modulate TLR4-dependent cytokine production. While these results may have broader implications for the role of TLR5 in the innate immune response in melioidosis and other conditions, further studies of the mechanisms underlying these observations are required.

Comparative accuracy of typhoid diagnostic tools: A Bayesian latent-class network analysis

by Paul Arora, Kristian Thorlund, Darren R. Brenner, Jason R. Andrews


Typhoid fevers are infections caused by the bacteria Salmonella enterica serovar Typhi (Salmonella Typhi) and Paratyphi A, B and C (Salmonella Paratyphi). Approximately 17.8 million incident cases of typhoid fever occur annually, and incidence is highest in children. The accuracy of current diagnostic tests of typhoid fever is poorly understood. We aimed to determine the comparative accuracy of available tests for the pediatric population.


We first conducted a systematic literature review to identify studies that compared diagnostic tests for typhoid fever in children (aged ≤15 years) to blood culture results. We applied a Bayesian latent-class extension to a network meta-analysis model. We modelled known diagnostic properties of bone marrow culture and the relationship between bone marrow and blood culture as informative priors in a Bayesian framework. We tested sensitivities for the proportion of negative blood samples that were false as well as bone marrow sensitivity and specificity.


We found 510 comparisons from 196 studies and 57 specific to the pediatric population. IgM-based tests outperformed their IgG-based counterparts for ELISA and Typhidot tests. The lateral flow IgG test performed comparatively well with 92% sensitivity (72% to 98% across scenario analyses) and 94% specificity. The most sensitive test of those investigated for the South Asian pediatric population was the Reverse Passive Hemagglutination Assay with 96% sensitivity (98% - 100% across scenario analyses). Adding a Widal slide test to other typhoid diagnostics did not substantially improve diagnostic performance beyond the single test alone, however, a lateral flow-based IgG rapid test combined with the typhoid/paratyphoid (TPT) assay yielded improvements in sensitivity without substantial declines in specificity and was the best performing combination test in this setting.


In the pediatric population, lateral-flow IgG, TPT and Reverse Passive Hemagglutination tests had high diagnostic accuracy compared to other diagnostics. Combinations of tests may provide a feasible option to increase diagnostic sensitivity. South Asia has the most informed set of data on typhoid diagnostic testing accuracy, and the evidence base in other important regions needs to be expanded.

A computational method for the identification of Dengue, Zika and Chikungunya virus species and genotypes

by Vagner Fonseca, Pieter J. K. Libin, Kristof Theys, Nuno R. Faria, Marcio R. T. Nunes, Maria I. Restovic, Murilo Freire, Marta Giovanetti, Lize Cuypers, Ann Nowé, Ana Abecasis, Koen Deforche, Gilberto A. Santiago, Isadora C. de Siqueira, Emmanuel J. San, Kaliane C. B. Machado, Vasco Azevedo, Ana Maria Bispo-de Filippis, Rivaldo Venâncio da Cunha, Oliver G. Pybus, Anne-Mieke Vandamme, Luiz C. J. Alcantara, Tulio de Oliveira

In recent years, an increasing number of outbreaks of Dengue, Chikungunya and Zika viruses have been reported in Asia and the Americas. Monitoring virus genotype diversity is crucial to understand the emergence and spread of outbreaks, both aspects that are vital to develop effective prevention and treatment strategies. Hence, we developed an efficient method to classify virus sequences with respect to their species and sub-species (i.e. serotype and/or genotype). This ArboTyping tool provides an easy-to-use software implementation of this new method and was validated on a large dataset assessing the classification performance with respect to whole-genome sequences and partial-genome sequences. Available online:

Endoparasites and vector-borne pathogens in dogs from Greek islands: Pathogen distribution and zoonotic implications

by Anastasia Diakou, Angela Di Cesare, Simone Morelli, Mariasole Colombo, Lenaig Halos, Giulia Simonato, Androniki Tamvakis, Frederic Beugnet, Barbara Paoletti, Donato Traversa

The present study investigated the presence of endo- and ecto-parasites, and vector-borne pathogens, in dogs from four islands of Greece. A total of 200 (123 owned and 77 sheltered) dogs were examined with different microscopic, serological and molecular methods. Of the examined dogs, 130 (65%) were positive for one or more parasites and/or vector-borne pathogens. The most common zoonotic intestinal helminths recorded were Ancylostomatidae (12.5%) and Toxocara canis (3.5%). Ninety-three dogs (46.5%) seroreacted to Rickettsia conorii. Twenty-two (11%) of them were also PCR positive and 7 (3.5%) showed corpuscles suggestive of Rickettsia spp. on the blood smears. Nineteen dogs (9.5%) were seropositive for Ehrlichia canis, three of them being also PCR positive. Dogs positive for Anaplasma phagocytophilum-Anaplasma platys (1%), Dirofilaria immitis (0.5%) and Babesia canis (0.5%) were also found. Fleas and ticks were recorded in 53 (26.5%) and 50 (25%) dogs, respectively, and all specimens were identified as Ctenocephalides felis felis and Rhipicephalus sanguineus sensu latu. Binary multiple univariate Generalized Linear Models were used to investigate factors and clinical signs related to the recorded positivity, while the association of specific signs with the pathogens was evaluated using tests of independence. Knowledge of occurrence and impact of zoonotic parasites and vector-borne pathogens in dog populations is crucial to prevent the infection in animals and people, and to control the risk of spreading of these pathogens in endemic and non-endemic areas.

PCR-RFLP analyses of <i>Leishmania</i> species causing cutaneous and mucocutaneous leishmaniasis revealed distribution of genetically complex strains with hybrid and mito-nuclear discordance in Ecuador

by Hirotomo Kato, Eduardo A. Gomez, Chisato Seki, Hayato Furumoto, Luiggi Martini-Robles, Jenny Muzzio, Manuel Calvopiña, Lenin Velez, Makoto Kubo, Ahmed Tabbabi, Daisuke S. Yamamoto, Yoshihisa Hashiguchi

PCR-Restriction Fragment Length Polymorphism (RFLP) analyses targeting multiple nuclear genes were established for the simple and practical identification of Leishmania species without using expensive equipment. This method was applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador, which have been identified at the species level by the kinetoplast cytochrome b (cyt b) gene sequence analysis, and the results obtained by the two analyses were compared. Although most results corresponded between the two analyses, PCR-RFLP analyses revealed distribution of hybrid strains between Leishmania (Viannia) guyanensis and L. (V.) braziliensis and between L. (V.) guyanensis and L. (V.) panamensis, of which the latter was firstly identified in Ecuador. Moreover, unexpected parasite strains having the kinetoplast cyt b gene of L. (V.) braziliensis and nuclear genes of L. (V.) guyanensis, L. (V.) panamensis, or a hybrid between L. (V.) guyanensis and L. (V.) panamensis were identified. This is the first report of the distribution of a protozoan parasite having mismatches between kinetoplast and nuclear genes, known as mito-nuclear discordance. The result demonstrated that genetically complex Leishmania strains are present in Ecuador. Since genetic exchanges such as hybrid formation were suggested to cause higher pathogenicity in Leishmania and may be transmitted by more species of sand flies, further country-wide epidemiological studies on clinical symptoms, as well as transmissible vectors, will be necessary.

Characterization of a non-sexual population of <i>Strongyloides stercoralis</i> with hybrid 18S rDNA haplotypes in Guangxi, Southern China

by Siyu Zhou, Xiaoyin Fu, Pei Pei, Marek Kucka, Jing Liu, Lili Tang, Tingzheng Zhan, Shanshan He, Yingguang Frank Chan, Christian Rödelsperger, Dengyu Liu, Adrian Streit

Strongyloidiasis is a much-neglected but sometimes fatal soil born helminthiasis. The causing agent, the small intestinal parasitic nematode Strongyloides stercoralis can reproduce sexually through the indirect/heterogonic life cycle, or asexually through the auto-infective or the direct/homogonic life cycles. Usually, among the progeny of the parasitic females both, parthenogenetic parasitic (females only) and sexual free-living (females and males) individuals, are present simultaneously. We isolated S. stercoralis from people living in a village with a high incidence of parasitic helminths, in particular liver flukes (Clonorchis sinensis) and hookworms, in the southern Chinese province Guangxi. We determined nuclear and mitochondrial DNA sequences of individual S. stercoralis isolated from this village and from close by hospitals and we compared these S. stercoralis among themselves and with selected published S. stercoralis from other geographic locations. For comparison, we also analyzed the hookworms present in the same location. We found that, compared to earlier studies of S. stercoralis populations in South East Asia, all S. stercoralis sampled in our study area were very closely related, suggesting a recent common source of infection for all patients. In contrast, the hookworms from the same location, while all belonging to the species Necator americanus, showed rather extensive genetic diversity even within host individuals. Different from earlier studies conducted in other geographic locations, almost all S. stercoralis in this study appeared heterozygous for different sequence variants of the 18S rDNA hypervariable regions (HVR) I and IV. In contrast to earlier investigations, except for three males, all S. stercoralis we isolated in this study were infective larvae, suggesting that the sampled population reproduces predominantly, if not exclusively through the clonal life cycles. Consistently, whole genome sequencing of individual worms revealed higher heterozygosity than reported earlier for likely sexual populations of S. stercoralis. Elevated heterozygosity is frequently associated with asexual clonal reproduction.

Hindgut microbiota in laboratory-reared and wild <i>Triatoma infestans</i>

by Andreea Waltmann, Alexandra C. Willcox, Sujata Balasubramanian, Katty Borrini Mayori, Sandra Mendoza Guerrero, Renzo S. Salazar Sanchez, Jeffrey Roach, Carlos Condori Pino, Robert H. Gilman, Caryn Bern, Jonathan J. Juliano, Michael Z. Levy, Steven R. Meshnick, Natalie M. Bowman

Triatomine vectors transmit Trypanosoma cruzi, the etiological agent of Chagas disease in humans. Transmission to humans typically occurs when contaminated triatomine feces come in contact with the bite site or mucosal membranes. In the Southern Cone of South America, where the highest burden of disease exists, Triatoma infestans is the principal vector for T. cruzi. Recent studies of other vector-borne illnesses have shown that arthropod microbiota influences the ability of infectious agents to colonize the insect vector and transmit to the human host. This has garnered attention as a potential control strategy against T. cruzi, as vector control is the main tool of Chagas disease prevention. Here we characterized the microbiota in T. infestans feces of both wild-caught and laboratory-reared insects and examined the relationship between microbial composition and T. cruzi infection using highly sensitive high-throughput sequencing technology to sequence the V3-V4 region of the 16S ribosomal RNA gene on the MiSeq Illumina platform. We collected 59 wild (9 with T. cruzi infection) and 10 lab-reared T. infestans (4 with T. cruzi infection) from the endemic area of Arequipa, Perú. Wild T. infestans had greater hindgut bacterial diversity than laboratory-reared bugs. Microbiota of lab insects comprised a subset of those identified in their wild counterparts, with 96 of the total 124 genera also observed in laboratory-reared insects. Among wild insects, variation in bacterial composition was observed, but time and location of collection and development stage did not explain this variation. T. cruzi infection in lab insects did not affect α- or β-diversity; however, we did find that the β-diversity of wild insects differed if they were infected with T. cruzi and identified 10 specific taxa that had significantly different relative abundances in infected vs. uninfected wild T. infestans (Bosea, Mesorhizobium, Dietzia, and Cupriavidus were underrepresented in infected bugs; Sporosarcina, an unclassified genus of Porphyromonadaceae, Nestenrenkonia, Alkalibacterium, Peptoniphilus, Marinilactibacillus were overrepresented in infected bugs). Our findings suggest that T. cruzi infection is associated with the microbiota of T. infestans and that inferring the microbiota of wild T. infestans may not be possible through sampling of T. infestans reared in the insectary.

Seasonal patterns of dengue fever in rural Ecuador: 2009-2016

by Rachel Sippy, Diego Herrera, David Gaus, Ronald E. Gangnon, Jonathan A. Patz, Jorge E. Osorio

Season is a major determinant of infectious disease rates, including arboviruses spread by mosquitoes, such as dengue, chikungunya, and Zika. Seasonal patterns of disease are driven by a combination of climatic or environmental factors, such as temperature or rainfall, and human behavioral time trends, such as school year schedules, holidays, and weekday-weekend patterns. These factors affect both disease rates and healthcare-seeking behavior. Seasonality of dengue fever has been studied in the context of climatic factors, but short- and long-term time trends are less well-understood. With 2009–2016 medical record data from patients diagnosed with dengue fever at two hospitals in rural Ecuador, we used Poisson generalized linear modeling to determine short- and long-term seasonal patterns of dengue fever, as well as the effect of day of the week and public holidays. In a subset analysis, we determined the impact of school schedules on school-aged children. With a separate model, we examined the effect of climate on diagnosis patterns. In the first model, the most important predictors of dengue fever were annual sinusoidal fluctuations in disease, long-term trends (as represented by a spline for the full study duration), day of the week, and hospital. Seasonal trends showed single peaks in case diagnoses, during mid-March. Compared to the average of all days, cases were more likely to be diagnosed on Tuesdays (risk ratio (RR): 1.26, 95% confidence interval (CI) 1.05–1.51) and Thursdays (RR: 1.25, 95% CI 1.02–1.53), and less likely to be diagnosed on Saturdays (RR: 0.81, 95% CI 0.65–1.01) and Sundays (RR: 0.74, 95% CI 0.58–0.95). Public holidays were not significant predictors of dengue fever diagnoses, except for an increase in diagnoses on the day after Christmas (RR: 2.77, 95% CI 1.46–5.24). School schedules did not impact dengue diagnoses in school-aged children. In the climate model, important climate variables included the monthly total precipitation, an interaction between total precipitation and monthly absolute minimum temperature, an interaction between total precipitation and monthly precipitation days, and a three-way interaction between minimum temperature, total precipitation, and precipitation days. This is the first report of long-term dengue fever seasonality in Ecuador, one of few reports from rural patients, and one of very few studies utilizing daily disease reports. These results can inform local disease prevention efforts, public health planning, as well as global and regional models of dengue fever trends.