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Detection of Zika virus in mouse mammary gland and breast milk

11 February 2019 - 10:00pm

by Jose Angel Regla-Nava, Karla M. Viramontes, Teodora Vozdolska, Anh-Thy Huynh, Tom Villani, Graeme Gardner, Michael Johnson, Pamela J. Ferro, Sujan Shresta, Kenneth Kim

Clinical reports of Zika Virus (ZIKV) RNA detection in breast milk have been described, but evidence conflicts as to whether this RNA represents infectious virus. We infected post-parturient AG129 murine dams deficient in type I and II interferon receptors with ZIKV. ZIKV RNA was detected in pup stomach milk clots (SMC) as early as 1 day post maternal infection (dpi) and persisted as late as 7 dpi. In mammary tissues, ZIKV replication was demonstrated by immunohistochemistry in multiple cell types including cells morphologically consistent with myoepithelial cells. No mastitis was seen histopathologically. In the SMC and tissues of the nursing pups, no infectious virus was detected via focus forming assay. However, serial passages of fresh milk supernatant yielded infectious virus, and immunohistochemistry showed ZIKV replication protein associated with degraded cells in SMC. These results suggest that breast milk may contain infectious ZIKV. However, breast milk transmission (BMT) does not occur in this mouse strain that is highly sensitive to ZIKV infection. These results suggest a low risk for breast milk transmission of ZIKV, and provide a platform for investigating ZIKV entry into milk and mechanisms which may prevent or permit BMT.

Comparing the performance of urine and copro-antigen detection in evaluating <i>Opisthorchis viverrini</i> infection in communities with different transmission levels in Northeast Thailand

8 February 2019 - 10:00pm

by Chanika Worasith, Chompunoot Wangboon, Kunyarat Duenngai, Nadda Kiatsopit, Kulthida Kopolrat, Anchalee Techasen, Jiraporn Sithithaworn, Narong Khuntikeo, Watcharin Loilome, Nisana Namwat, Puangrat Yongvanit, Elizabeth J. Carlton, Paiboon Sithithaworn

To combat and eventually eliminate the transmission of the liver fluke Opisthorchis viverrini, an accurate and practical diagnostic test is required. A recently established urine antigen detection test using monoclonal antibody-based enzyme-immunosorbent assay (mAb-ELISA) has shown promise due to its high diagnostic accuracy and the use of urine in place of fecal samples. To further test the utility of this urine assay, we performed a cross sectional study of 1,043 people in 3 opisthorchiasis endemic communities in northeast Thailand by applying urine antigen detection together with copro-antigen detection methods. The quantitative formalin-ethyl acetate concentration technique (FECT) was concurrently performed as a reference method. The prevalence of O. viverrini determined by urine antigen detection correlated well with that by copro-antigen detection and both methods showed 10–15% higher prevalence than FECT. Within the fecal negative cases by FECT, 29% and 43% were positive by urine and copro-antigen detection, respectively. The prevalence and intensity profiles determined by antigen detection and FECT showed similar patterns of increasing trends of infection with age. The concentration of antigen measured in urine showed a positive relationship with the concentration of copro-antigen, both of which were positively correlated with fecal egg counts. The data observed in this study indicate that urine antigen detection had high diagnostic accuracy and was in concordance with copro-antigen detection. Due to the ease and noninvasiveness of sample collection, the urine assay has high potential for clinical diagnosis as well as population screening in the program for the control and elimination of opisthorchiasis.

Etiology and severity of diarrheal diseases in infants at the semiarid region of Brazil: A case-control study

8 February 2019 - 10:00pm

by Aldo A. M. Lima, Domingos B. Oliveira, Josiane S. Quetz, Alexandre Havt, Mara M. G. Prata, Ila F. N. Lima, Alberto M. Soares, José Q. Filho, Noélia L. Lima, Pedro H. Q. S. Medeiros, Ana K. S. Santos, Herlice N. Veras, Rafhaella N. D. G. Gondim, Rafaela C. Pankov, Mariana D. Bona, Francisco A. P. Rodrigues, Renato A. Moreira, Ana C. O. M. Moreira, Marcelo Bertolini, Luciana R. Bertolini, Vicente J. F. Freitas, Eric R. Houpt, Richard L. Guerrant

Background

Diarrheal diseases are an important cause of morbidity and mortality among children in developing countries. We aimed to study the etiology and severity of diarrhea in children living in the low-income semiarid region of Brazil.

Methodology

This is a cross-sectional, age-matched case-control study of diarrhea in children aged 2–36 months from six cities in Brazil’s semiarid region. Clinical, epidemiological, and anthropometric data were matched with fecal samples collected for the identification of enteropathogens.

Results

We enrolled 1,200 children, 596 cases and 604 controls. By univariate analysis, eight enteropathogens were associated with diarrhea: Norovirus GII (OR 5.08, 95% CI 2.10, 12.30), Adenovirus (OR 3.79, 95% CI 1.41, 10.23), typical enteropathogenic Escherichia coli (tEPEC), (OR 3.28, 95% CI 1.39, 7.73), enterotoxigenic E. coli (ETEC LT and ST producing toxins), (OR 2.58, 95% CI 0.99, 6.69), rotavirus (OR 1.91, 95% CI 1.20, 3.02), shiga toxin-producing E. coli (STEC; OR 1.77, 95% CI 1.16, 2.69), enteroaggregative E. coli (EAEC), (OR 1.45, 95% CI 1.16, 1.83) and Giardia spp. (OR 1.39, 95% CI 1.05, 1.84). By logistic regression of all enteropathogens, the best predictors of diarrhea were norovirus, adenovirus, rotavirus, STEC, Giardia spp. and EAEC. A high diarrhea severity score was associated with EAEC.

Conclusions

Six enteropathogens: Norovirus, Adenovirus, Rotavirus, STEC, Giardia spp., and EAEC were associated with diarrhea in children from Brazil’s semiarid region. EAEC was associated with increased diarrhea severity.

Evaluation of a class of isatinoids identified from a high-throughput screen of human kinase inhibitors as anti-Sleeping Sickness agents

8 February 2019 - 10:00pm

by Dana M. Klug, Rosario Diaz-Gonzalez, Guiomar Pérez-Moreno, Gloria Ceballos-Pérez, Raquel García-Hernández, Veronica Gomez-Pérez, Luis Miguel Ruiz-Pérez, Domingo I. Rojas-Barros, Francisco Gamarro, Dolores González-Pacanowska, María S. Martínez-Martínez, Pilar Manzano, Lori Ferrins, Conor R. Caffrey, Miguel Navarro, Michael P. Pollastri

New treatments are needed for neglected tropical diseases (NTDs) such as Human African trypanosomiasis (HAT), Chagas disease, and schistosomiasis. Through a whole organism high-throughput screening campaign, we previously identified 797 human kinase inhibitors that grouped into 59 structural clusters and showed activity against T. brucei, the causative agent of HAT. We herein report the results of further investigation of one of these clusters consisting of substituted isatin derivatives, focusing on establishing structure-activity and -property relationship scope. We also describe their in vitro absorption, distribution, metabolism, and excretion (ADME) properties. For one isatin, NEU-4391, which offered the best activity-property profile, pharmacokinetic parameters were measured in mice.

Serological evidence of infection with dengue and Zika viruses in horses on French Pacific Islands

7 February 2019 - 10:00pm

by Cécile Beck, Isabelle Leparc-Goffart, Denise Desoutter, Estelle Debergé, Hervé Bichet, Steeve Lowenski, Marine Dumarest, Gaelle Gonzalez, Camille Migné, Jessica Vanhomwegen, Stéphan Zientara, Benoit Durand, Sylvie Lecollinet

New Caledonia and French Polynesia are areas in which arboviruses circulate extensively. A large serological survey among horses from New Caledonia and French Polynesia was carried out to investigate the seroprevalence of flaviviruses in the horse population. Here, 293 equine sera samples were screened for flaviviruses using a competitive enzyme-linked immunosorbent assay (cELISA). The positive sera were then confirmed using a flavivirus-specific microsphere immunoassay (MIA) and seroneutralization tests. This serosurvey showed that 16.6% (27/163) and 30.8% (40/130) of horses were positive for cELISA tests in New Caledonia and French Polynesia, respectively, but the MIA technique, targeting only flaviviruses causing neuro-invasive infections in humans and horses (i.e. West Nile virus [WNV], Japanese encephalitis virus [JEV] and tick-borne encephalitis virus [TBEV]), showed negative results for more than 85% (57/67) of the cELISA-positive animals. Seroneutralization tests with the main flaviviruses circulating in the South Pacific revealed that 6.1% (10/163; confidence interval [95% CI] 3.0%-11.0%) of sera in New Caledonia and 7.7% (10/130; 95% CI 3.8%-13.7%) in French Polynesia were positive for dengue virus serotype 1 (DENV1) and 4.3% (7/163; 95% CI 1.7%-8.6%) in New Caledonia and 15.4% (20/130, 95% CI 9.7%-22.8%) in French Polynesia were found positive for Zika virus (ZIKV). Seroprevalence of the JEV and WNV flaviviruses on the 293 samples from both island groups were comparatively much lower (less than 2%). This seroprevalence study in the horse population shows that horses can be infected with dengue and Zika viruses and that these infections lead to seroconversions in horses. The consequences of these infections in horses and their role in ZIKV and DENV epidemiological cycles are two issues that deserve further investigation.

Inward rectifier potassium (Kir) channels mediate salivary gland function and blood feeding in the lone star tick, <i>Amblyomma americanum</i>

7 February 2019 - 10:00pm

by Zhilin Li, Kevin R. Macaluso, Lane D. Foil, Daniel R. Swale

Background

Tick feeding causes extreme morbidity and mortality to humans through transmission of pathogens and causes severe economic losses to the agricultural industry by reducing livestock yield. Salivary gland secretions are essential for tick feeding and thus, reducing or preventing saliva secretions into the vertebrate host is likely to reduce feeding and hinder pathogen life cycles. Unfortunately, the membrane physiology of tick salivary glands is underexplored and this gap in knowledge limits the development of novel therapeutics for inducing cessation of tick feeding.

Methodology

We studied the influence of inward rectifier potassium (Kir) channel subtypes to the functional capacity of the isolated tick salivary gland through the use of a modified Ramsay assay. The secreted saliva was subsequently used for quantification of the elemental composition of the secreted saliva after the glands were exposed to K+ channel modulators as a measure of osmoregulatory capacity. Lastly, changes to blood feeding behavior and mortality were measured with the use of a membrane feeding system.

Principal findings

In this study, we characterized the fundamental role of Kir channel subtypes in tick salivary gland function and provide evidence that pharmacological inhibition of these ion channels reduces the secretory activity of the salivary gland of Amblyomma americanum. The reduced secretory capacity of the salivary gland was directly correlated with a dramatic reduction of blood ingestion during feeding. Further, exposure to small-molecule modulators of Kir channel subtypes induced mortality to ticks that is likely resultant from an altered osmoregulatory capacity.

Conclusions

Our data contribute to understanding of tick salivary gland function and could guide future campaigns aiming to develop chemical or reverse vaccinology technologies to reduce the worldwide burden of tick feeding and tick-vectored pathogens.

Knowledge, attitudes and practices with regard to schistosomiasis prevention and control: Two cross-sectional household surveys before and after a Community Dialogue intervention in Nampula province, Mozambique

7 February 2019 - 10:00pm

by Christian Rassi, Sandrine Martin, Kirstie Graham, Monica Anna de Cola, Celine Christiansen-Jucht, Lauren E. Smith, Ercílio Jive, Anna E. Phillips, James N. Newell, Marilia Massangaie

Background

The Community Dialogue Approach is a promising social and behaviour change intervention, which has shown potential for improving health seeking behaviour. To test if this approach can strengthen prevention and control of schistosomiasis at community level, Malaria Consortium implemented a Community Dialogue intervention in four districts of Nampula province, Mozambique, between August 2014 and September 2015.

Methodology/Principal findings

Cross-sectional household surveys were conducted before (N = 791) and after (N = 792) implementation of the intervention to assess its impact on knowledge, attitudes and practices at population level. At both baseline and endline, awareness of schistosomiasis was high at over 90%. After the intervention, respondents were almost twice as likely to correctly name a risk behaviour associated with schistosomiasis (baseline: 18.02%; endline: 30.11%; adjusted odds ratio: 1.91; 95% confidence interval: 1.14–2.58). Increases were also seen in the proportion of people who knew that schistosomiasis can be spread by infected persons and who could name at least one correct transmission route (baseline: 25.74%; endline: 32.20%; adjusted odds ratio: 1.36; 95% confidence interval: 1.01–1.84), those who knew that there is a drug that treats the disease (baseline: 29.20%, endline: 47.55%; adjusted odds ratio: 2.19; 95% confidence interval: 1.67–2.87) and those who stated that they actively protect themselves from the disease and cited an effective behaviour (baseline: 40.09%, endline: 59.30%; adjusted odds ratio: 2.14; 95% confidence interval: 1.40–3.28). The intervention did not appear to lead to a reduction in misconceptions. In particular, the belief that the disease is sexually transmitted continued to be widespread.

Conclusions/Significance

Given its overall positive impact on knowledge and behaviour at population level, Community Dialogue can play an important role in schistosomiasis prevention and control. The intervention could be further strengthened by better enabling communities to take suitable action and linking more closely with community governance structures and health system programmes.

Very severe tungiasis in Amerindians in the Amazon lowland of Colombia: A case series

7 February 2019 - 10:00pm

by Hollman Miller, Jovana Ocampo, Alvaro Ayala, Julian Trujillo, Hermann Feldmeier

Background

Tungiasis is a parasitic skin disease caused by penetrating female sand fleas. By nature, tungiasis is a self-limiting infection. However, in endemic settings re-infection is the rule and parasite load gradually accumulates over time. Intensity of infection and degree of morbidity are closely related.

Methodology/principal findings

This case series describes the medical history, the clinical pathology, the socio-economic and the environmental characteristics of very severe tungiasis in five patients living in traditional Amerindian communities in the Amazon lowland of Colombia. Patients had between 400 and 1,300 penetrated sand fleas. The feet were predominantly affected, but clusters of embedded sand fleas also occurred at the ankles, the knees, the elbows, the hands, the fingers and around the anus. The patients were partially or totally immobile. Patients 1 and 3 were cachectic, patient 2 presented severe malnutrition. Patient 3 needed a blood transfusion due to severe anemia. All patients showed a characteristic pattern of pre-existing medical conditions and culture-dependent behavior facilitating continuous re-infection. In all cases intradomiciliary transmission was very likely.

Conclusion/significance

Although completely ignored in the literature, very severe tungiasis occurs in settings where patients do not have access to health care and are stricken in a web of pre-existing illness, poverty and neglect. If not treated, very severe tungiasis may end in a fatal disease course.

Protective immunity by an engineered DNA vaccine for Mayaro virus

7 February 2019 - 10:00pm

by Hyeree Choi, Sagar B. Kudchodkar, Emma L. Reuschel, Kanika Asija, Piyush Borole, Michelle Ho, Krzysztof Wojtak, Charles Reed, Stephanie Ramos, Nathen E. Bopp, Patricia V. Aguilar, Scott C. Weaver, J. Joseph Kim, Laurent Humeau, Pablo Tebas, David B. Weiner, Kar Muthumani

Mayaro virus (MAYV) of the genus alphavirus is a mosquito-transmitted emerging infectious disease that causes an acute febrile illness, rash, headaches, and nausea that may turn into incapacitating, persistent arthralgias in some victims. Since its discovery in Trinidad in 1954, cases of MAYV infection have largely been confined there and to the northern countries of South America, but recently, MAYV cases have been reported in some island nations in the Caribbean Sea. Accompanying these reports is evidence that new vectors, including Aedes spp. mosquitos, recently implicated in the global spread of Zika and chikungunya viruses, are competent for MAYV transmission, which, if true, could facilitate the spread of MAYV beyond its current range. Despite its status as an emerging virus, there are no licensed vaccines to prevent MAYV infection nor therapeutics to treat it. Here, we describe the development and testing of a novel DNA vaccine, scMAYV-E, that encodes a synthetically-designed consensus MAYV envelope sequence. In vivo electroporation-enhanced immunization of mice with this vaccine induced potent humoral responses including neutralizing antibodies as well as robust T-cell responses to multiple epitopes in the MAYV envelope. Importantly, these scMAYV-E-induced immune responses protected susceptible mice from morbidity and mortality following a MAYV challenge.

Strategies to increase adoption of animal vaccines by smallholder farmers with focus on neglected diseases and marginalized populations

7 February 2019 - 10:00pm

by Meritxell Donadeu, Nick Nwankpa, Bernadette Abela-Ridder, Baptiste Dungu

Background

Most smallholder farmers (SHFs) and marginalized populations (MPs) in Africa, Asia, and Latin America depend on livestock for their livelihoods. However, significant numbers of these animals do not achieve their potential, die due to disease, or transmit zoonotic diseases. Existing vaccines could prevent and control some of these diseases, but frequently the vaccines do not reach SHFs, especially MPs, making it necessary for specific vaccine adoption strategies.

Principal findings

Several strategies that have the potential to increase the adoption of animal vaccines by SHFs and MPs have been identified depending on the type of vaccines involved. The strategies differed depending on whether the vaccines were aimed at diseases that cause economic losses, government-controlled diseases, or neglected diseases. The adoption of vaccines for neglected diseases presents a major challenge, because they are mostly for zoonotic diseases that produce few or no clinical signs in the animals, making it more difficult for the farmers to appreciate the value of the vaccines.Strategies can be aimed at increasing the availability of quality vaccines, so that they are produced in sufficient quantity, or aimed at increasing access and demand by SHFs and/or MPs. Some of the strategies to increase vaccine adoption might not provide a definite solution but might facilitate vaccine uptake by decreasing barriers. These strategies are varied and include technical considerations, policy components, involvement by the private sector (local and international), and innovation.

Conclusions

Several strategies with the potential to reduce livestock morbidity and mortality, or prevent zoonoses in SHFs communities and MPs through vaccination, require the involvement of donors and international organisations to stimulate and facilitate sustainable adoption. This is especially the case for neglected zoonotic diseases. Support for national and regional vaccine manufacturers is also required, especially for vaccines against diseases of interest only in the developing world and public goods.

Reprogramming of <i>Trypanosoma cruzi</i> metabolism triggered by parasite interaction with the host cell extracellular matrix

6 February 2019 - 10:00pm

by Eliciane C. Mattos, Gisele Canuto, Nubia C. M. Varón, Rubens D. M. Magalhães, Thomas W. M. Crozier, Douglas J. Lamont, Marina F. M. Tavares, Walter Colli, Michael A. J. Ferguson, Maria Júlia M. Alves

Trypanosoma cruzi, the etiological agent of Chagas’ disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.

The validity of diagnostic cut-offs for commercial and in-house scrub typhus IgM and IgG ELISAs: A review of the evidence

4 February 2019 - 10:00pm

by Kartika Saraswati, Meghna Phanichkrivalkosil, Nicholas P. J. Day, Stuart D. Blacksell

Background

Scrub typhus is a neglected tropical disease that causes acute febrile illness. Diagnosis is made based upon serology, or detection of the causative agent–Orientia tsutsugamushi–using PCR or in vitro isolation. The enzyme-linked immunosorbent assay (ELISA) is an objective and reproducible means of detecting IgM or IgG antibodies. However, lack of standardization in ELISA methodology, as well as in the choice of reference test with which the ELISA is compared, calls into question the validity of cut-offs used in diagnostic accuracy studies and observational studies.

Methodology/Principal findings

A PubMed search and manual screening of reference lists identified 46 studies that used ELISA antibody cut-offs to diagnose scrub typhus patients, 22 of which were diagnostic accuracy studies. Overall, 22 studies (47.8%) provided little to no explanation as to how the ELISA cut-off was derived, and 7 studies (15.2%) did not even state the cut-off used. Variation was seen locally in reference standards used, in terms of both the diagnostic test and cut-off titer. Furthermore, with the exception of studies using ELISAs manufactured by InBios, there was no standardization of the selection of antigenic strains. As a result, no consensus was found for determining a cut-off, ELISA methodology, or for a single value diagnostic cut-off.

Conclusions/Significance

We have concluded that there is a lack of consensus in the determination of a cut-off. We recommend interpreting the results from these studies with caution. Further studies will need to be performed at each geographic location to determine region-specific cut-offs, taking into consideration background antibody levels to discriminate true disease from healthy individuals.

Water-induced strong protection against acute exposure to low subzero temperature of adult <i>Aedes albopictus</i>

4 February 2019 - 10:00pm

by Meichun Zhang, Dongjing Zhang, Yongjun Li, Qiang Sun, Qin Li, Yali Fan, Yu Wu, Zhiyong Xi, Xiaoying Zheng

As an important vector of dengue and Zika, Aedes albopictus has been the fastest spreading invasive mosquitoes in the world over the last 3–4 decades. Cold tolerance is important for survival and expansion of insects. Ae. albopictus adults are generally considered to be cold-intolerant that cannot survive at subzero temperature. However, we found that Ae. albopictus could survive for several hours’ exposure to -9 to -19 oC so long as it was exposed with water. Median lethal time (LT50) of Ae. albopictus exposed to -15 and -19 oC with water increased by more than 100 times compared to those exposed to the same subzero temperature without water. This phenomenon also existed in adult Aedes aegypti and Culex quinquefasciatus. Ae. albopictus female adults which exposed to low subzero temperature at -9 oC with water had similar longevity and reproductive capacity to those of females without cold exposure. Cold exposure after a blood meal also have no detrimental impact on survival capacity of female adult Ae. albopictus compared with those cold exposed without a blood meal. Moreover, our results showed that rapid cold hardening (RCH) was induced in Ae. albopictus during exposing to low subzero temperature with water. Both the RCH and the relative high subzero temperature of water immediate after cold exposure might provide this strong protection against low subzero temperature. The molecular basis of water-induced protection for Ae. albopictus might refer to the increased glycerol during cold exposure, as well as the increased glucose and hsp70 during recovery from cold exposure. Our results suggested that the water-induced strong protection against acute decrease of air temperature for adult mosquitoes might be important for the survival and rapid expansion of Ae. albopictus.

Comparative fitness of West Nile virus isolated during California epidemics

4 February 2019 - 10:00pm

by Gabriella Worwa, Andra A. Hutton, Aaron C. Brault, William K. Reisen

West Nile virus (WNV) has been circulating in California since its first detection in 2003, causing repeated outbreaks affecting public, wildlife and veterinary health. Epidemics of WNV are difficult to predict due to the multitude of factors influencing transmission dynamics among avian and mosquito hosts. Typically, high levels of WNV amplification are required for outbreaks to occur, and therefore associated viral strains may exhibit enhanced virulence and mortality in competent bird species resulting in increased mosquito infection prevalence. In our previous study, most WNV isolates made from California during 2007–08 showed increased fitness when competed in House Finches (HOFI, Haemorhous mexicanus) and Culex tarsalis Coquillett mosquitoes against COAV997-5nt, a genetically marked recombinant virus derived from a 2003 California strain. Herein, we evaluated the competitive fitness of WNV strains isolated during California epidemics in 2004, 2005, 2007, 2011 and 2012 against COAV997-5nt. These outbreak isolates did not produce elevated mortality in HOFIs, but replicated more efficiently than did COAV997-5nt based on quantification of WNV RNA copies in sera, thereby demonstrating increased competitive fitness. Oral co-infections in Cx. tarsalis resulted in similar virus-specific infection and transmission rates, indicating that outbreak isolates did not have a fitness advantage over COAV997-5nt. Collectively, WNV isolates from outbreaks demonstrated relatively greater avian, but not vector, replicative fitness compared to COAV997-5nt, similar to previously characterized non-outbreak isolates of WNV. Our results indicated that ecological rather than viral factors may facilitate WNV amplification to outbreak levels, but monitoring viral phenotypes through competitive fitness studies may provide insight into altered replication and transmission potential among emerging WNV strains.

Experimental Zika virus infection of Jamaican fruit bats (<i>Artibeus jamaicensis</i>) and possible entry of virus into brain via activated microglial cells

4 February 2019 - 10:00pm

by Ashley Malmlov, Collin Bantle, Tawfik Aboellail, Kaitlyn Wagner, Corey L. Campbell, Miles Eckley, Nunya Chotiwan, Rebecca C. Gullberg, Rushika Perera, Ronald Tjalkens, Tony Schountz

The emergence of Zika virus (ZIKV) in the New World has led to more than 200,000 human infections. Perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with Guillain-Barré syndrome (GBS). ZIKV is transmitted to humans by Aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal Aedes sp. mosquitos and non-human primates. In the 1950s and ‘60s, several bat species were shown to be naturally and experimentally susceptible to ZIKV with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected the brain. Because of ZIKV emergence in the Americas, we sought to determine susceptibility of Jamaican fruit bats (Artibeus jamaicensis), one of the most common bats in the New World. Bats were inoculated with ZIKV PRVABC59 but did not show signs of disease. Bats held to 28 days post-inoculation (PI) had detectable antibody by ELISA and viral RNA was detected by qRT-PCR in the brain, saliva and urine in some of the bats. Immunoreactivity using polyclonal anti-ZIKV antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. Tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular Leydig cells. The virus likely localized to the brain via infection of Iba1+ macrophage/microglial cells. Jamaican fruit bats, therefore, may be a useful animal model for the study of ZIKV infection. This work also raises the possibility that bats may have a role in Zika virus ecology in endemic regions, and that ZIKV may pose a wildlife disease threat to bat populations.

Genomic instability at the locus of sterol C24-methyltransferase promotes amphotericin B resistance in <i>Leishmania</i> parasites

4 February 2019 - 10:00pm

by Andrew W. Pountain, Stefan K. Weidt, Clément Regnault, Paul A. Bates, Anne M. Donachie, Nicholas J. Dickens, Michael P. Barrett

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3’ untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern.

Rapid detection of <i>Mycobacterium ulcerans</i> with isothermal recombinase polymerase amplification assay

1 February 2019 - 10:00pm

by Michael Frimpong, Hubert Senanu Ahor, Ahmed Abd El Wahed, Bernadette Agbavor, Francisca Naana Sarpong, Kenneth Laing, Mark Wansbrough-Jones, Richard Odame Phillips

Background

Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease.

Methodology and principal findings

A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84–100), whiles the sensitivity was 88% (95% CI, 77–95).

Conclusion

The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.

Identification of French Guiana sand flies using MALDI-TOF mass spectrometry with a new mass spectra library

1 February 2019 - 10:00pm

by Agathe Chavy, Cécile Nabet, Anne Cécile Normand, Arthur Kocher, Marine Ginouves, Ghislaine Prévot, Thiago Vasconcelos dos Santos, Magalie Demar, Renaud Piarroux, Benoît de Thoisy

Phlebotomine sand flies are insects that are highly relevant in medicine, particularly as the sole proven vectors of leishmaniasis. Accurate identification of sand fly species is an essential prerequisite for eco-epidemiological studies aiming to better understand the disease. Traditional morphological identification is painstaking and time-consuming, and molecular methods for extensive screening remain expensive. Recent studies have shown that matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for rapid and cost-effective identification of arthropod vectors, including sand flies. The aim of this study was to validate the use of MALDI-TOF MS for the identification of Northern Amazonian sand flies. We constituted a MALDI-TOF MS reference database comprising 29 species of sand flies that were field-collected in French Guiana, which are expected to cover many of the more common species of the Northern Amazonian region, including known vectors of leishmaniasis. Carrying out a blind test, all the sand flies tested (n = 157) with a log (score) threshold greater than 1.7 were correctly identified at the species level. We confirmed that MALDI-TOF MS protein profiling is a useful tool for the study of sand flies, including neotropical species, known for their great diversity. An application that includes the spectra generated here will be available to the scientific community in the near future via an online platform.

Growing evidence of <i>Plasmodium vivax</i> across malaria-endemic Africa

31 January 2019 - 10:00pm

by Katherine A. Twohig, Daniel A. Pfeffer, J. Kevin Baird, Ric N. Price, Peter A. Zimmerman, Simon I. Hay, Peter W. Gething, Katherine E. Battle, Rosalind E. Howes

Effective malaria control strategies require an accurate understanding of the epidemiology of locally transmitted Plasmodium species. Compared to Plasmodium falciparum infection, Plasmodium vivax has a lower asexual parasitaemia, forms dormant liver-stages (hypnozoites), and is more transmissible. Hence, treatment and diagnostic policies aimed exclusively at P. falciparum are far less efficient against endemic P. vivax. Within sub-Saharan Africa, malaria control programmes justly focus on reducing the morbidity and mortality associated with P. falciparum. However, the recent emphasis on malaria elimination and increased accessibility of more sensitive diagnostic tools have revealed greater intricacies in malaria epidemiology across the continent. Since 2010, the number of studies identifying P. vivax endemic to Africa has expanded considerably, with 88 new scientific reports published since a review of evidence in 2015, approximately doubling the available data. There is evidence of P. vivax in all regions of Africa, apparent from infected vectors, clinical cases, serological indicators, parasite prevalence, exported infections, and P. vivax-infected Duffy-negative individuals. Where the prevalence of microscopic parasitaemia is low, a greater proportion of P. vivax infections were observed relative to P. falciparum. This evidence highlights an underlying widespread presence of P. vivax across all malaria-endemic regions of Africa, further complicating the current practical understanding of malaria epidemiology in this region. Thus, ultimate elimination of malaria in Africa will require national malaria control programmes to adopt policy and practice aimed at all human species of malaria.

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