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Distribution and breeding sites of <i>Aedes aegypti</i> and <i>Aedes albopictus</i> in 32 urban/peri-urban districts of Mozambique: implication for assessing the risk of arbovirus outbreaks

12 September 2018 - 9:00pm

by Ana Paula Abílio, Gastão Abudasse, Ayubo Kampango, Baltazar Candrinho, Salomão Sitoi, Jacinta Luciano, Dário Tembisse, Samira Sibindy, António Paulo Gouveia de Almeida, Gabriela Azambuja Garcia, Mariana Rocha David, Rafael Maciel-de-Freitas, Eduardo Samo Gudo

Background

Aedes-borne arboviruses have emerged as an important public health problem worldwide and, in Mozambique, the number of cases and its geographical spread have been growing. However, information on the occurrence, distribution and ecology of Aedes aegypti and Ae. albopictus mosquitoes remain poorly known in the country.

Methods

Between March and April 2016, a cross-sectional study was conducted in 32 districts in Mozambique to determine the distribution and breeding sites of Ae. aegypti and Ae. albopictus. Larvae and pupae were collected from a total of 2,807 water-holding containers using pipette, dipper, funnel and sweeping procedures, depending on the container type and location. Both outdoor and indoor water-holding containers were inspected. The immature forms were reared to adults and the identifications of the mosquito species was carried out with a stereomicroscope using a taxonomic key.

Results

Aedes aegypti was found in every district sampled, while Ae. albopictus was only found in Moatize district, situated in Tete Province in the central part of the country. Six hundred and twenty-eight of 2,807 (22.4%) containers were positive for Ae. aegypti but only one (0.03%) was positive for Ae. albopictus. The Container Index (CI) of Aedes was highest in densely populated suburban areas of the central region (260/604; 43.0%), followed by suburban areas in northern areas (228/617; 36.9%) whilst the lowest proportion was found in urbanized southern areas (140/1586; 8.8%). The highest CI of Aedes was found in used tires (448/1268; 35.3%), cement tanks (20/62; 32.3%) and drums (21/95; 22.1%).

Conclusion

Data from our study showed that Ae. aegypti is present nation-wide, since it occurred in every sampled district, whilst Ae. albopictus had a limited distribution. Therefore, the risk of transmission of dengue and chikungunya is likely to have been underestimated in Mozambique. This study highlights the need for the establishment of a national entomological surveillance program for Aedes spp. in Mozambique in order to gain a better understanding about vector bionomics and to support the development of informed effective vector control strategies.

Tick-borne zoonoses in the Order Rickettsiales and Legionellales in Iran: A systematic review

11 September 2018 - 9:00pm

by Faham Khamesipour, Gabriel O. Dida, Douglas N. Anyona, Mostafa Razavi, Ehsan Rakhshandehroo

Background

Tick-borne zoonoses in the Order Rickettsiales and Legionellales cause infections that often manifest as undifferentiated fevers that are not easy to distinguish from other causes of acute febrile illnesses clinically. This is partly attributed to difficulty in laboratory confirmation since convalescent sera, specific diagnostic reagents, and the required expertise may not be readily available. As a result, a number of tick-borne zoonoses are underappreciated resulting in unnecessary morbidity, mortality and huge economic loses. In Iran, a significant proportion of human infectious diseases are tick-borne, with anecdotal evidence suggesting that tick-borne zoonoses are widespread but underreported in the country. Epidemiological review is therefore necessary to aid in the effective control and prevention of tick-borne zonooses in Iran. The aim of this review is to provide an in-depth and comprehensive overview of anaplasmosis, ehrlichiosis, spotted fever group rickettsioses and coxiellosis in Iran.

Methods

Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, all relevant publications on tick-borne zoonoses in the Order Rickettsiales and Legionellales in Iran were searched using a number of search terms. The search was confined to authentic resources from repositories of popular data bases among them PubMed, Web of Science, Google Scholar, Science Direct, SpringerLink and SCOPUS. The search items included peer reviewed journals, books and book chapters published between 1996 and 2017.

Results

A total of 1 205 scientific publications and reports were sourced, of which 63 met the search criteria and were reviewed. Of the 63 articles reviewed, 36 (57.1%) reported on coxiellosis, 15 (23.8%) on anaplasmosis, 11 (17.5%) on ehrlichiosis and 1(1.6%) on spotted fever group rickettsiae in a large scale study involving four countries, among them Iran. The existence of tick-borne pathogens in the Order Rickettsiales and Legionellales was confirmed by molecular, serological and microscopic techniques conducted on samples obtained from sheep, cattle, goats, camels, poultry, animal products (milk and eggs), dogs, ticks and even human subjects in different parts of the country; pointing to a countrywide distribution.

Discussion

Based on the review, coxiellosis, anaplasmosis, ehrlichiosis, and SFG rickettsiae can be categorized as emerging tick-borne zoonotic diseases in Iran given the presence of their causiative agents (C. burnetii, A. phagocytophilum, A. marginale, A. bovis, A. ovis, A. central, E. canis, E. ewingii, E. chaffeensis and R. conorii) collectively reported in a variety of domestic animals, animal products, arthropods and human beings drawn from 22 provinces in Iran.

Conclusion

Given the asymptomatic nature of some of these zoonoses, there is a high likelihood of silent transmission to humans in many parts of the country, which should be considered a public health concern. Presently, information on the transmission intensity of tick-borne zoonoses caused by pathogens in the Order Rickettsiales and Legionellales to humans and its public health impact in Iran is scanty.

Comparison of individual and pooled diagnostic examination strategies during the national mapping of soil-transmitted helminths and <i>Schistosoma mansoni</i> in Ethiopia

10 September 2018 - 9:00pm

by Gemechu Tadesse Leta, Mike French, Pierre Dorny, Jozef Vercruysse, Bruno Levecke

Background

Laboratory-based studies have highlighted that pooling stool and urine samples can reduce costs and diagnostic burden without a negative impact on the ability to estimate the intensity of soil-transmitted helminth (STH, Ascaris lumbricoides, Trichuris trichiura and hookworms) and schistosome infections (Schistosoma mansoni and S. haematobium). In this study, we compare individual and pooled stool examination strategies in a programmatic setting.

Methods

Stool samples were collected from 2,650 children in 53 primary schools in Amhara Regional State, Ethiopia, during the national mapping of STHs and schistosome infections. Eggs of STHs and S. mansoni were quantified in both individual and pooled samples (pools were made from 10 individual samples) using a single Kato-Katz smear.

Principal findings

A pooled diagnostic examination strategy provided comparable estimates of infection intensity with higher fecal egg count (expressed in eggs per gram of stool (EPG)) than those based on individual strategy (Ascaris: 45.1 EPG vs. 93.9, p = 0.03; Trichuris: 1.8 EPG vs. 2.1 EPG, p = 0.95; hookworms: 17.5 EPG vs. 28.5 EPG, p = 0.18; S. mansoni: 1.6 EPG vs. 3.4 EPG, p = 0.02), but had lower sensitivity (Ascaris: 90.0% vs. 55.0%; Trichuris: 91.7% vs. 16.7%; hookworms: 92.6% vs. 61.8%; S. mansoni: 100% vs. 51.7%, p < 0.001). A pooled approach resulted in a ~70% reduction in time required for sample testing, but reduced total operational costs by only ~11%.

Conclusions/Significance

A pooled approach holds promise for the rapid assessment of intensity of helminth infections in a programmatic setting, but it is not major cost-saving strategy. Further investigation is required to determine when and how pooling can be utilized. Such work should also include validation of statistical methods to estimate prevalence based on pooling samples. Finally, the comparison of operational costs across different scenarios of national program management will help determine whether pooling is indeed worthwhile considering.

Feasibility and initial outcomes of a multifaceted prevention programme of melioidosis in diabetic patients in Ubon Ratchathani, northeast Thailand

6 September 2018 - 9:00pm

by Pornpan Suntornsut, Wipada Chaowagul, Wilasinee Thongklang, Thidarat Phosri, Nicholas P. J. Day, Susan Michie, Direk Limmathurotsakul

Background

Melioidosis is an infection caused by Burkholderia pseudomallei, a Gram-negative bacillus found in soil and water. Diabetes mellitus is the most important risk factor for melioidosis. The recommendations for disease prevention include avoiding direct contact with soil and water, and drinking only boiled or bottled water.

Methods

A prospective intervention study was conducted to evaluate the feasibility and behavioural outcomes of a multifaceted prevention programme for melioidosis. Participants were diabetic adults in Ubon Ratchathani, northeast Thailand. Ten behavioural support groups consisting of 6 to 10 participants per group were conducted. Twelve behaviour change techniques were used: information about health consequences, credible source, adding objects to the environment, reconstructing the physical environment, instruction on how to perform a behaviour, demonstration of the behaviour, commitment, prompts/cues, self-monitoring of behaviour, goal setting, feedback on behaviour, and social support, and their feasibilities evaluated.

Results

There were 70 participants, of median age 59 years and 52 (74%) were female. Participants found the intervention beneficial, interesting and engaging. Participants indicated that they liked to watch videos with information about melioidosis delivered by local doctors and patients who survived melioidosis, and videos showing use of over-the-knee boots by local farmers. Participants felt engaged in the sessions that trialed protective gear and that made calendars with individual photographs and self-pledges as a reminder tool. The proportions of participants reporting that they always wore boots while working in rice fields increased from 30% (10/33) to 77% (28/37, p = 0.04), and that they drank only boiled or bottle water increased from 43% (30/70) to 86% (59/69, p<0.001) at 6 months post intervention.

Conclusion

The programme is highly acceptable to participants, and can support behaviour change. Policy makers should consider implementing the programme in areas where melioidosis is endemic. Making calendars with individual photographs and self-pledges as a reminder tool could be powerful in behaviour change interventions, and further research on this component is needed.

Typhoid fever in Santiago, Chile: Insights from a mathematical model utilizing venerable archived data from a successful disease control program

6 September 2018 - 9:00pm

by Jillian S. Gauld, Hao Hu, Daniel Klein, Myron M. Levine

Typhoid fever is endemic in many developing countries. In the early 20th century, newly industrializing countries including the United States successfully controlled typhoid as water treatment (chlorination/sand filtration) and improved sanitation became widespread. Enigmatically, typhoid remained endemic through the 1980s in Santiago, Chile, despite potable municipal water and widespread household sanitation. Data were collected across multiple stages of endemicity and control in Santiago, offering a unique resource for gaining insight into drivers of transmission in modern settings. We developed an individual-based mathematical model of typhoid transmission, with model components including distinctions between long-cycle and short-cycle transmission routes. Data used to fit the model included the prevalence of chronic carriers, seasonality, longitudinal incidence, and age-specific distributions of typhoid infection and disease. Our model captured the dynamics seen in Santiago across endemicity, vaccination, and environmental control. Both vaccination and diminished exposure to seasonal amplified long-cycle transmission contributed to the observed declines in typhoid incidence, with the vaccine estimated to elicit herd effects. Vaccines are important tools for controlling endemic typhoid, with even limited coverage eliciting herd effects in this setting. Removing the vehicles responsible for amplified long-cycle transmission and assessing the role of chronic carriers in endemic settings are additional key elements in designing programs to achieve accelerated control of endemic typhoid.

Evaluation of the recombinant antigens B2t and 2B2t, compared with hydatid fluid, in IgG-ELISA and immunostrips for the diagnosis and follow up of CE patients

6 September 2018 - 9:00pm

by Ana Hernández-González, Carlos Sánchez-Ovejero, Raúl Manzano-Román, María González Sánchez, José Manuel Delgado, Teresa Pardo-García, Francisco Soriano-Gálvez, Okan Akhan, Carmen M. Cretu, Kamenna Vutova, Francesca Tamarozzi, Mara Mariconti, Enrico Brunetti, Ambra Vola, Massimo Fabiani, Adriano Casulli, Mar Siles-Lucas

Cystic echinococcosis (CE) is one of the most widespread helminthic zoonoses and is caused by the tapeworm Echinococcus granulosus complex. CE diagnosis and monitoring primarily rely on imaging techniques, complemented by serology. This is usually approached by the detection of IgG antibodies against hydatid fluid (HF), but the use of this heterogeneous antigenic mixture results in a variable percentage of false positive and negative results, and has shown to be useless for follow-up due to the long persistence of anti-HF antibodies in cured patients. To improve test performances and standardization, a number of recombinant antigens mainly derived from HF have been described, among them the B2t and 2B2t antigens. The performance of these antigens in the diagnosis and follow up of patients with CE has been so far evaluated on a limited number of samples. Here, we evaluated the performances of tests based on B2t and 2B2t recombinant antigens compared to HF in IgG-ELISA and immunochromatography (IC) for the diagnosis and follow-up of patients with CE in a retrospective cohort study. A total of 721 serum samples were collected: 587 from 253 patients with CE diagnosed by ultrasonography (US), 42 from patients with alveolar echinococcosis and 92 from healthy donors from Salamanca (Spain). The highest overall sensitivity was obtained with HF in ELISA (85.5%), followed by IC containing HF and 2B2t-HF (83.0% and 78.2%, respectively). The lowest sensitivity was obtained with B2t and 2B2t in ELISA (51.8%). The highest specificity was obtained with IC containing 2B2t-HF (100%), and the lowest with HF-ELISA (78.0%). The lowest cross-reactivity with sera from patients with alveolar echinococcosis was detected with the recombinant antigens in ELISA (9.5% - 16.7%) and the highest with the HF-IC (64.3%). The results of B2t and 2B2t-ELISA were influenced by cyst stage, as classified by US according to the WHO-Informal Working Group on Echinococcosis (WHO-IWGE), with low sensitivity for inactive (CE4 and CE5) cysts, and by the drug treatment, with higher sensitivity in patients after drug treatment compared with patients not subjected to drug treatment. The two recombinant antigens in ELISA provided promising results for monitoring patients in follow-up, although their use is limited to patients with positive serology against them at the beginning of the follow-up. Potential biological reasons behind the low sensitivity of the recombinant antigens and possible strategies to enhance the performance of CE serology are discussed.

Phylogenomic analysis unravels evolution of yellow fever virus within hosts

6 September 2018 - 9:00pm

by Chen Chen, Dong Jiang, Ming Ni, Jing Li, Zhihai Chen, Jingyuan Liu, Hanhui Ye, Gary Wong, Wei Li, Yuanyuan Zhang, Beibei Wang, Yuhai Bi, Danying Chen, Ping Zhang, Xuesen Zhao, Yaxian Kong, Weifeng Shi, Pengcheng Du, Gengfu Xiao, Juncai Ma, George F. Gao, Jie Cui, Fujie Zhang, Wenjun Liu, Xiaochen Bo, Ang Li, Hui Zeng, Di Liu

The yellow fever virus (YFV) recently reemerged in the large outbreaks in Africa and Brazil, and the first imported patients into Asia have recalled the concerns of YFV evolution. Here we show phylogenomics of YFV with serial clinical samples of the 2016 YFV infections. Phylogenetics exhibited that the 2016 strains were close to Angola 1971 strains and only three amino acid changes presented new to other lineages. Deep sequencing of viral genomes discovered 101 intrahost single nucleotide variations (iSNVs) and 234 single nucleotide polymorphisms (SNPs). Analysis of iSNV distribution and mutated allele frequency revealed that the coding regions were under purifying selection. Comparison of the evolutionary rates estimated by iSNV and SNP showed that the intrahost rate was ~2.25 times higher than the epidemic rate, and both rates were higher than the long-term YFV substitution rate, as expected. In addition, the result also hinted that short viremia duration of YFV might further hinder the evolution of YFV.

Burden of leishmaniasis in Brazil and federated units, 1990-2016: Findings from Global Burden of Disease Study 2016

6 September 2018 - 9:00pm

by Juliana Maria Trindade Bezerra, Valdelaine Etelvina Miranda de Araújo, David Soeiro Barbosa, Francisco Rogerlândio Martins-Melo, Guilherme Loureiro Werneck, Mariângela Carneiro

Background

The study presents estimates for the burden of visceral leishmaniasis (VL) and cutaneous and mucocutaneous leishmaniasis (CML) in Brazil and its 27 federated units using data from the Global Burden of Disease Study (GBD) 2016.

Methodology

We report the incidence, years of life lost (YLL), years lived with disability (YLD), and disability-adjusted life years (DALY) for leishmaniasis in Brazil from 1990 to 2016. The metrics are presented as age-standardized rates per 100,000 inhabitants with their respective uncertainty intervals (95%UI) and relative percentages of change.

Principal findings

The age-standardized incidence rate of leishmaniasis decreased 48.5% from 1990 (71.0, 95%UI 24.3–150.7) to 2016 (36.5, 95%UI 24.7–50.9), whereas the age-standardized DALY increased 83.6% over the studied period from 12.2 (95%UI 7.9–18.8) to 22.4 (95%UI 13.3–36.2). The age-standardized incidence rate and YLL for VL increased by 52.9% and 108% from 1990 to 2016, respectively. Considering CML, the age-standardized incidence rate and YLD decreased by 51% and 31.8% respectively for the same period. For VL, similar profiles for male and female were observed, with YLL and DALY increasing over time; with males presenting slightly higher values. The highest YLL rates were among "under 1-year old" children, which increased 131.2% from 1990 to 2016. Regarding CML, the highest values of YLD and DALY were verified among males, and YLD values showed a similar profile, with rates increasing with age. The VL burden increased in some states in the Northeast and Southeast regions and decreased for CML in some Northern states.

Conclusion

The increase of VL burden over the study period might be associated with the difficulties in controlling the disease spread. Information regarding the weight of VL and CML, including the death and disability tolls that they cause, highlights the impact of these neglected diseases on public health and the importance of effective prevention and treatment.

Human rabies in Côte d'Ivoire 2014-2016: Results following reinforcements to rabies surveillance

6 September 2018 - 9:00pm

by Issaka Tiembré, Anaïs Broban, Joseph Bénié, Mathilde Tetchi, Sophie Druelles, Maïna L’Azou

In Côte d’Ivoire, rabies is endemic and remains largely uncontrolled. The numbers of human exposures and rabies cases are unknown and are probably much higher than reported. Data on human rabies cases are collected by the National Institute of Public Health (NIPH) Anti-rabies Center in Abidjan through a network of 28 NIPH local units, which cover the population of the entire country. During 2014, the NIPH initiated a program to reinforce the human rabies surveillance system in those 28 NIPH local units, with specific goals of improving the infrastructure, training, communication, and government involvement. Here, we report the progress and findings during 2014–2016. The reinforced system recorded 50 cases of human rabies (15–18 cases/year; annual incidence = 0.06−0.08 per 100,000) and more than 30,000 animal exposures (annual incidence = 41.8−48.0 per 100,000). Almost one-half of the human rabies cases were in children ≤15 years old. All were fatal and dog bites were the most common route by which rabies virus was transmitted. In the 32 cases where samples of sufficient quality for analysis were available, rabies was confirmed by reverse transcription-polymerase chain reaction RT-PCR. Post-exposure prophylaxis with rabies vaccine was administered to all animal exposure victims presenting at the NIPH local units, although only about 57% completed the full immunization schedule. All available reports were provided by the NIPH local units, indicating effective communication between them and the NIPH Anti-rabies Center. These findings indicate that the reinforcements resulted in highly specific detection of human rabies, provided detailed epidemiological data about these cases, and improved estimates of animal exposure numbers. These represent substantial advances, but further improvements to the surveillance system are needed to increase disease awareness and capture cases that are currently missed by the system. In the future, better communication between local health centers and the NIPH units, surveillance at the local health center level, and increased veterinarian engagement will help provide a more complete picture of the rabies burden in Côte d’Ivoire.

Improving systematic rabies surveillance in Cameroon: A pilot initiative and results for 2014-2016

6 September 2018 - 9:00pm

by Casimir Ledoux Sofeu, Anaïs Broban, Amadou Njifou Njimah, Jean Blaise Momo, Serge Alain Sadeuh-Mba, Sophie Druelles, Maïna L’Azou, Mathurin Cyrille Tejiokem

Canine rabies is endemic in Cameroon, but human rabies exposures and cases are likely underreported because of inadequate surveillance. In 2014, the surveillance network in the West region of Cameroon was reinforced by introducing a new anti-rabies center, a framework for data collection and evaluation, provisions for sample collecting and laboratory confirmation, and training for health professionals. The objective of this observational cohort study was to describe the incidence and characteristics of reported exposures and human and animal rabies cases following this reinforcement of the existing rabies surveillance system. The surveillance network consisted of local, regional, and national health and veterinary authorities in 11 of the 20 West region districts, and was completely integrated within the existing national rabies surveillance network. Animal exposures and suspected rabies exposures, the suspected rabid animals involved, and laboratory confirmation of human and animal rabies cases were recorded in a centralized information database. Between January 2014 and June 2016, the network recorded 1340 animal exposure cases for an overall incidence rate of 38.2 animal exposures per 100,000 people, four confirmed rabies-positive animals, and one confirmed human rabies case out of four clinically suspected cases. In contrast, 62 animal exposures and an overall incidence rate of 6.1 exposures per 100,000 people were reported for the West region districts not participating in the reinforced surveillance. Of the 925 animal exposure victims for whom a detailed case report form was completed, 703 were considered to be at risk of rabies and only 428 (61%) of these received any post-exposure prophylaxis in the form of rabies vaccine. Obstacles encountered within the network included low rates of animal sample submission and animal follow-up by veterinarians. Reinforced rabies surveillance in the West region of Cameroon has provided the most accurate estimate of the region’s disease and exposure burdens to date, and indicates that animal exposures are substantially underreported. The reinforced network also signaled that greater access to post-exposure prophylaxis is needed. Integration of regions not covered by the surveillance network and efforts to improve engagement of veterinary services will be needed to reveal the true burden of rabies in Cameroon.

Bolstering human rabies surveillance in Africa is crucial to eliminating canine-mediated rabies

6 September 2018 - 9:00pm

by Anaïs Broban, Mathurin C. Tejiokem, Issaka Tiembré, Sophie Druelles, Maïna L’Azou

Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

4 September 2018 - 9:00pm

by Ines Martin-Martin, Azadeh Aryan, Claudio Meneses, Zach N. Adelman, Eric Calvo

Background

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis.

Methodology/Principal findings

Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed.

Conclusion and significance

The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.

Evaluation of Auramine O staining and conventional PCR for leprosy diagnosis: A comparative cross-sectional study from Ethiopia

4 September 2018 - 9:00pm

by Selfu Girma, Charlotte Avanzi, Kidist Bobosha, Kassu Desta, Munir H. Idris, Philippe Busso, Yohannes Tsegaye, Shimelis Nigusse, Tsegaye Hailu, Stewart T. Cole, Abraham Aseffa

Background

Diagnosis of leprosy mainly relies on clinical examination due to the inconsistent sensitivity and poor reproducibility of the current laboratory tests. Utilisation of alternative methods to the standard Ziehl Neelsen (ZN), Fite-Faraco (FF) and Haematoxylin and Eosin (H&E) staining procedures may eventually improve leprosy diagnosis.

Methodology/Principal findings

In this comparative study, the performance of the fluorescent Auramine O (AO) staining and polymerase chain reaction (PCR) was assessed with different skin samples using a combination of ZN, FF and H&E staining as the gold standard. AO, ZN, FF, H&E and PCR tests were performed on slit skin smears (SSS) and/or punch biopsies collected from 141 clinically confirmed leprosy cases and 28 non-leprosy skin samples. DNA was extracted from punch biopsies using two different methods with or without mechanical lysis.Sensitivities were 87.6%, 59.3% and 77% for H&E, ZN and FF, respectively, whereas it reached 65.5% and 77.9% for AO in SSS and tissue sections and 91.1% for PCR in tissue samples. Morover, samples with low bacillary index, sensitivity of AO staining (61.8%) was similar to FF (60%, p>0.05) and lower than PCR (86.6%, p<0.05). Sensitivity of PCR also increased (96.8%, p<0.05) when mechanical lysis was used during DNA extraction compared to enzymatic treatment alone (84.6%).

Conclusions/Significance

Our results showed that for diagnostic purposes, analysis of skin section is more sensitive than SSS, especially for samples with low bacillary load. AO staining on SSS and tissue sections was not significantly better than other routine diagnostic tests but considerably more user friendly. The sensitivity of PCR was higher than current standard methods and increased when combined with more efficient DNA extraction using mechanical and chemical lysis. Therefore, we recommend AO staining for the diagnosis of leprosy in lower health facilities such as health centres and district hospitals and PCR diagnosis at referral level and research centres.

Mosquito excreta: A sample type with many potential applications for the investigation of Ross River virus and West Nile virus ecology

31 August 2018 - 9:00pm

by Ana L. Ramírez, Sonja Hall-Mendelin, Stephen L. Doggett, Glen R. Hewitson, Jamie L. McMahon, Scott A. Ritchie, Andrew F. van den Hurk

Background

Emerging and re-emerging arthropod-borne viruses (arboviruses) cause human and animal disease globally. Field and laboratory investigation of mosquito-borne arboviruses requires analysis of mosquito samples, either individually, in pools, or a body component, or secretion such as saliva. We assessed the applicability of mosquito excreta as a sample type that could be utilized during studies of Ross River and West Nile viruses, which could be applied to the study of other arboviruses.

Methodology/Principal findings

Mosquitoes were fed separate blood meals spiked with Ross River virus and West Nile virus. Excreta was collected daily by swabbing the bottom of containers containing batches and individual mosquitoes at different time points. The samples were analyzed by real-time RT-PCR or cell culture enzyme immunoassay. Viral RNA in excreta from batches of mosquitoes was detected continuously from day 2 to day 15 post feeding. Viral RNA was detected in excreta from at least one individual mosquito at all timepoints, with 64% and 27% of samples positive for RRV and WNV, respectively. Excretion of viral RNA was correlated with viral dissemination in the mosquito. The proportion of positive excreta samples was higher than the proportion of positive saliva samples, suggesting that excreta offers an attractive sample for analysis and could be used as an indicator of potential transmission. Importantly, only low levels of infectious virus were detected by cell culture, suggesting a relatively low risk to personnel handling mosquito excreta.

Conclusions/Significance

Mosquito excreta is easily collected and provides a simple and efficient method for assessing viral dissemination, with applications ranging from vector competence experiments to complementing sugar-based arbovirus surveillance in the field, or potentially as a sample system for virus discovery.

Demonstration of efficient vertical and venereal transmission of dengue virus type-2 in a genetically diverse laboratory strain of <i>Aedes aegypti</i>

31 August 2018 - 9:00pm

by Irma Sánchez-Vargas, Laura C. Harrington, Jeffrey B. Doty, William C. Black 4th, Ken E. Olson

Aedes aegypti is the primary mosquito vector of dengue viruses (DENV; serotypes 1–4). Human-mosquito transmission cycles maintain DENV during epidemics but questions remain regarding how these viruses survive when human infections and vector abundance are minimal. Aedes mosquitoes can transmit DENV within the vector population through two alternate routes: vertical and venereal transmission (VT and VNT, respectively). We tested the efficiency of VT and VNT in a genetically diverse laboratory (GDLS) strain of Ae. aegypti orally infected with DENV2 (Jamaica 1409). We examined F1 larvae from infected females generated during the first and second gonotrophic cycles (E1 and E2) for viral envelope (E) antigen by amplifying virus in C6/36 cells and then performing an indirect immunofluorescence assay (IFA). RT-PCR/nested PCR analyses confirmed DENV2 RNA in samples positive by IFA. We observed VT of virus to larvae and adult male progeny and VNT of virus to uninfected virgin females after mating with males that had acquired virus by the VT route. We detected no DENV2 in 30 pools (20 larvae/pool) of F1 larvae following the first gonotrophic cycle, suggesting limited virus dissemination at 7 days post-infection. DENV2 was detected by IFA in 27 of 49 (55%) and 35 of 51 (68.6%) F1 larval pools (20 larvae/pool) from infected E2 females that received a second blood meal without virus at 10 or 21 days post-infection (E2-10d-F1 and E2-21-F1), respectively. The minimum filial infection rates by IFA for E2-10d-F1 and E2-21d-F1 mosquitoes were 1:36 and 1:29, respectively. The VNT rate from E2-10d-F1 males to virgin (uninfected) GDLS females was 31.6% (118 of 374) at 8 days post mating. Twenty one percent of VNT-infected females receiving a blood meal prior to mating had disseminated virus in their heads, suggesting a potential pathway for virus to re-enter the human-mosquito transmission cycle. This is the first report of VNT of DENV by male Ae. aegypti and the first demonstration of sexual transmission in Aedes by naturally infected males. Our results demonstrate the potential for VT and VNT of DENV in nature as mechanisms for virus maintenance during inter-epidemic periods.

Fine-scale GPS tracking to quantify human movement patterns and exposure to leptospires in the urban slum environment

31 August 2018 - 9:00pm

by Katharine A. Owers, Juliana Odetunde, Rosan Barbosa de Matos, Gielson Sacramento, Mayara Carvalho, Nivison Nery Jr, Federico Costa, Mitermayer G. Reis, James E. Childs, José E. Hagan, Peter J. Diggle, Albert I. Ko

Background

Human movement is likely an important risk factor for environmentally-transmitted pathogens. While epidemiologic studies have traditionally focused on household risk factors, individual movement data could provide critical additional information about risk of exposure to such pathogens. We conducted global positioning system (GPS) tracking of urban slum residents to quantify their fine-scale movement patterns and evaluate their exposures to environmental sources of leptospirosis transmission.

Methodology/Principal findings

We recruited participants from an ongoing cohort study in an urban slum in Brazil and tracked them for 24 hours at 30-second intervals. Among 172 subjects asked to participate in this cross-sectional study, 130 agreed to participate and 109 had good quality data and were included in analyses. The majority of recorded locations were near participant residences (87.7% within 50 meters of the house), regardless of age or gender. Similarly, exposure to environmental sources of leptospirosis transmission did not vary by age or gender. However, males, who have higher infection rates, visited a significantly larger area during the 24-hour period than did females (34,549m2 versus 22,733m2, p = 0.005). Four male participants had serologic evidence of Leptospira infection during the study period. These individuals had significantly larger activity spaces than uninfected males (61,310m2 vs 31,575m2, p = 0.006) and elevated exposure to rodent activity (p = 0.046) and trash deposits (p = 0.031).

Conclusions/Significance

GPS tracking was an effective tool for quantifying individual mobility in the complex urban slum environment and identifying risk exposures associated with that movement. This study suggests that in addition to source reduction, barrier interventions that reduce contact with transmission sources as slum residents move within their communities may be a useful prevention strategy for leptospirosis.

Barcoding blood meals: New vertebrate-specific primer sets for assigning taxonomic identities to host DNA from mosquito blood meals

30 August 2018 - 9:00pm

by Lawrence E. Reeves, Jennifer L. Gillett-Kaufman, Akito Y. Kawahara, Phillip E. Kaufman

The transmission dynamics of mosquito-vectored pathogens are, in part, mediated by mosquito host-feeding patterns. These patterns are elucidated using blood meal analysis, a collection of serological and molecular techniques that determine the taxonomic identities of the host animals from which blood meals are derived. Modern blood meal analyses rely on polymerase chain reaction (PCR), DNA sequencing, and bioinformatic comparisons of blood meal DNA sequences to reference databases. Ideally, primers used in blood meal analysis PCRs amplify templates from a taxonomically diverse range of vertebrates, produce a short amplicon, and avoid co-amplification of non-target templates. Few primer sets that fit these requirements are available for the cytochrome c oxidase subunit I (COI) gene, the species identification marker with the highest taxonomic coverage in reference databases. Here, we present new primer sets designed to amplify fragments of the DNA barcoding region of the vertebrate COI gene, while avoiding co-amplification of mosquito templates, without multiplexed or nested PCR. Primers were validated using host vertebrate DNA templates from mosquito blood meals of known origin, representing all terrestrial vertebrate classes, and field-collected mosquito blood meals of unknown origin. We found that the primers were generally effective in amplifying vertebrate host, but not mosquito DNA templates. Applied to the sample of unknown mosquito blood meals, > 98% (60/61) of blood meals samples were reliably identified, demonstrating the feasibility of identifying mosquito hosts with the new primers. These primers are beneficial in that they can be used to amplify COI templates from a diverse range of vertebrate hosts using standard PCR, thereby streamlining the process of identifying the hosts of mosquitoes, and could be applied to next generation DNA sequencing and metabarcoding approaches.

“Zika is everywhere”: A qualitative exploration of knowledge, attitudes and practices towards Zika virus among women of reproductive age in Iquitos, Peru

30 August 2018 - 9:00pm

by Caroline T. Weldon, Amy R. Riley-Powell, Ines M. Aguerre, Rosa A. Celis Nacimento, Amy C. Morrison, Richard A. Oberhelman, Valerie A. Paz-Soldan

Zika virus was reported in the rainforest city of Iquitos, Peru in 2016. The potential associations between Zika and fetal neurological disorders were reported extensively in the media regarding neighboring Brazil, and led to great concern about the impact Zika could have on people’s health in Iquitos when it arrived. The aim of this study was to explore the knowledge, attitudes, and preventative practices related to Zika virus and its transmission among women of childbearing age in Iquitos, Peru. Six focus group discussions with 46 women of ages 20–35 from an Iquitos district with confirmed Zika cases were conducted to explore: 1) knowledge of Zika transmission, its symptoms, and treatment, 2) attitudes regarding Zika, including perceptions of risk for and severity of Zika, and 3) preventative practices, including awareness of health promotion activities. Participants were knowledgeable about Zika symptoms and knew it was transmitted by mosquitoes, and about half had heard about the association between Zika and microcephaly, but most lacked knowledge about the associated neurological disorders in adults, its sexual transmission, and ways to prevent infection. They expressed concern for pregnant women exposed to the virus and the impact on the fetus. Participants felt at risk of contracting the Zika virus, yet had not changed preventive practices, possibly in part because their perception of the severity of this disease was low. This study reveals knowledge gaps that could be addressed via health promotion messages that might improve prevention practices to help community members protect themselves from Zika virus during this outbreak.

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of <i>Wolbachia</i> using a cellphone

30 August 2018 - 9:00pm

by Sanchita Bhadra, Timothy E. Riedel, Miguel A. Saldaña, Shivanand Hegde, Nicole Pederson, Grant L. Hughes, Andrew D. Ellington

Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.

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