RESUMO
BACKGROUND: Predicting when and where pathogens will emerge is difficult, yet, as shown by the recent Ebola and Zika epidemics, effective and timely responses are key. It is therefore crucial to transition from reactive to proactive responses for these pathogens. To better identify priorities for outbreak mitigation and prevention, we developed a cohesive framework combining disparate methods and data sources, and assessed subnational pandemic potential for four viral haemorrhagic fevers in Africa, Crimean-Congo haemorrhagic fever, Ebola virus disease, Lassa fever, and Marburg virus disease. METHODS: In this multistage analysis, we quantified three stages underlying the potential of widespread viral haemorrhagic fever epidemics. Environmental suitability maps were used to define stage 1, index-case potential, which assesses populations at risk of infection due to spillover from zoonotic hosts or vectors, identifying where index cases could present. Stage 2, outbreak potential, iterates upon an existing framework, the Index for Risk Management, to measure potential for secondary spread in people within specific communities. For stage 3, epidemic potential, we combined local and international scale connectivity assessments with stage 2 to evaluate possible spread of local outbreaks nationally, regionally, and internationally. FINDINGS: We found epidemic potential to vary within Africa, with regions where viral haemorrhagic fever outbreaks have previously occurred (eg, western Africa) and areas currently considered non-endemic (eg, Cameroon and Ethiopia) both ranking highly. Tracking transitions between stages showed how an index case can escalate into a widespread epidemic in the absence of intervention (eg, Nigeria and Guinea). Our analysis showed Chad, Somalia, and South Sudan to be highly susceptible to any outbreak at subnational levels. INTERPRETATION: Our analysis provides a unified assessment of potential epidemic trajectories, with the aim of allowing national and international agencies to pre-emptively evaluate needs and target resources. Within each country, our framework identifies at-risk subnational locations in which to improve surveillance, diagnostic capabilities, and health systems in parallel with the design of policies for optimal responses at each stage. In conjunction with pandemic preparedness activities, assessments such as ours can identify regions where needs and provisions do not align, and thus should be targeted for future strengthening and support. FUNDING: Paul G Allen Family Foundation, Bill & Melinda Gates Foundation, Wellcome Trust, UK Department for International Development.
Assuntos
Febres Hemorrágicas Virais/epidemiologia , Pandemias , África/epidemiologia , Surtos de Doenças/estatística & dados numéricos , Epidemias/estatística & dados numéricos , Humanos , Pandemias/estatística & dados numéricos , Medição de RiscoRESUMO
BACKGROUND: Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack. CONCLUSIONS/SIGNIFICANCE: Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.
Assuntos
Aedes/parasitologia , Brugia/isolamento & purificação , Culex/parasitologia , Mosquitos Vetores/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Brugia/classificação , Brugia/genética , DNA de Helmintos/genética , Filariose Linfática/parasitologia , Filariose Linfática/transmissão , Humanos , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Sensibilidade e Especificidade , Wuchereria bancrofti/genética , Wuchereria bancrofti/isolamento & purificaçãoRESUMO
BACKGROUND: The monitoring and evaluation of lymphatic filariasis (LF) has largely relied on the detection of antigenemia and antibodies in human populations. Molecular xenomonitoring (MX), the detection of parasite DNA/RNA in mosquitoes, may be an effective complementary method, particularly for detecting signals in low-level prevalence areas where Culex is the primary mosquito vector. This paper investigated the application of a household-based sampling method for MX in Tamil Nadu, India. METHODS: MX surveys were conducted in 2010 in two evaluation units (EUs): 1) a hotspot area, defined as sites with community microfilaria prevalence ≥1%, and 2) a larger area that also encompassed the hotspots. Households were systematically selected using a sampling interval proportional to the number of households in the EU. Mosquito pools were collected and analyzed by real-time polymerase chain reaction (qPCR). Two independent samples were taken in each EU to assess reproducibility of results. Follow-up surveys were conducted in 2012. RESULTS: In 2010, the proportion of positive pools in the hotspot EU was 49.3% compared to 23.4% in the overall EU. In 2012, pool positivity was significantly reduced to 24.3% and 6.5%, respectively (p<0.0001). Pool positivity based on independent samples taken from each EU in 2010 and 2012 were not significantly different except for the hotspot EU in 2012 (p = 0.009). The estimated prevalence of infection in mosquitoes, measured by PoolScreen, declined from 2.2-2.7% in 2010 to 0.6-1.2% in 2012 in the hotspot area and from 0.9-1.1% to 0.2-0.3% in the larger area. CONCLUSIONS: The household-based sampling strategy for MX led to mostly reproducible results and supported the observed LF infection trends found in humans. MX has the potential to be a cost-effective, non-invasive monitoring and evaluation tool with sensitive detection of infection signals in low prevalence settings. Further investigation and application of this sampling strategy for MX are recommended to support its adoption as a standardized method for global LF elimination programs.
Assuntos
Controle de Doenças Transmissíveis/métodos , Culex/parasitologia , Filariose Linfática/epidemiologia , Wuchereria bancrofti/isolamento & purificação , Animais , DNA de Helmintos/isolamento & purificação , Filariose Linfática/prevenção & controle , Características da Família , Humanos , Índia/epidemiologia , Microfilárias/genética , Microfilárias/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Wuchereria bancrofti/genéticaRESUMO
We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samples collected in endemic areas in Egypt and Papua New Guinea. Although the two methods had comparable sensitivity for detecting filarial DNA in reference samples, real-time PCR was more sensitive than C-PCR in practice with field samples. Other advantages of real-time PCR include its high-throughput capacity and decreased risk of cross-contamination between test samples. We believe that real-time PCR has great potential as a tool for monitoring progress in large-scale filariasis elimination programs.
Assuntos
Anopheles/parasitologia , Culex/parasitologia , Filariose/diagnóstico , Wuchereria bancrofti/genética , Animais , Primers do DNA , DNA Bacteriano/análise , DNA de Protozoário/análise , Egito , Filariose/sangue , Filariose/transmissão , Humanos , Insetos Vetores/parasitologia , Papua Nova Guiné , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , RNA Ribossômico 16S/análise , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Wolbachia/genética , Wolbachia/isolamento & purificação , Wuchereria bancrofti/isolamento & purificaçãoRESUMO
Successful mass drug administration (MDA) campaigns have brought several countries near the point of Lymphatic Filariasis (LF) elimination. A diagnostic tool is needed to determine when the prevalence levels have decreased to a point that MDA campaigns can be discontinued without the threat of recrudescence. A six-country study was conducted assessing the performance of seven diagnostic tests, including tests for microfilariae (blood smear, PCR), parasite antigen (ICT, Og4C3) and antifilarial antibody (Bm14, PanLF, Urine SXP). One community survey and one school survey were performed in each country. A total of 8,513 people from the six countries participated in the study, 6,443 through community surveys and 2,070 through school surveys. Specimens from these participants were used to conduct 49,585 diagnostic tests. Each test was seen to have both positive and negative attributes, but overall, the ICT test was found to be 76% sensitive at detecting microfilaremia and 93% specific at identifying individuals negative for both microfilariae and antifilarial antibody; the Og4C3 test was 87% sensitive and 95% specific. We conclude, however, that the ICT should be the primary tool recommended for decision-making about stopping MDAs. As a point-of-care diagnostic, the ICT is relatively inexpensive, requires no laboratory equipment, has satisfactory sensitivity and specificity and can be processed in 10 minutes-qualities consistent with programmatic use. Og4C3 provides a satisfactory laboratory-based diagnostic alternative.
Assuntos
Anti-Helmínticos/uso terapêutico , Filariose Linfática/tratamento farmacológico , Wuchereria bancrofti , Adolescente , Adulto , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/urina , Criança , Pré-Escolar , Filariose Linfática/epidemiologia , Filariose Linfática/parasitologia , Feminino , Saúde Global , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prevalência , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: Detection of filarial DNA in mosquitoes by PCR cannot differentiate infective mosquitoes from infected mosquitoes. In order to evaluate transmission risk an assay is needed that can specifically detect infective L3 stage parasites. We now report the development of an assay that specifically detects the infective stage of Wuchereria bancrofti in mosquitoes. The assay detects an L3-activated mRNA transcript by reverse-transcriptase PCR (RT-PCR). METHODOLOGY/PRINCIPAL FINDINGS: W. bancrofti cuticle-related genes were selected using bioinformatics and screened as potential diagnostic target genes for L3 detection in mosquitoes. Expression profiles were determined using RT-PCR on RNA isolated from mosquitoes collected daily across a two-week period after feeding on infected blood. Conventional multiplex RT-PCR and real-time multiplex RT-PCR assays were developed using an L3-activated cuticlin transcript for L3 detection and a constitutively expressed transcript, tph-1, for 'any-stage' detection. CONCLUSIONS/SIGNIFICANCE: This assay can be used to simultaneously detect W. bancrofti infective stage larvae and 'any-stage' larvae in pooled vector mosquitoes. This test may be useful as a tool for assessing changes in transmission potential in the context of filariasis elimination programs.
Assuntos
Culicidae/parasitologia , Vetores de Doenças , Parasitologia/métodos , RNA de Helmintos/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Wuchereria bancrofti/isolamento & purificação , Animais , Sequência de Bases , Primers do DNA/genética , Larva , Dados de Sequência Molecular , RNA de Helmintos/genética , RNA Mensageiro/genética , Sensibilidade e Especificidade , Wuchereria bancrofti/genéticaRESUMO
Seven rounds of mass drug administration (MDA) have been administered in Leogane, Haiti, an area hyperendemic for lymphatic filariasis (LF). Sentinel site surveys showed that the prevalence of microfilaremia was reduced to <1% from levels as high as 15.5%, suggesting that transmission had been reduced. A separate 30-cluster survey of 2- to 4-year-old children was conducted to determine if MDA interrupted transmission. Antigen and antifilarial antibody prevalence were 14.3% and 19.7%, respectively. Follow-up surveys were done in 6 villages, including those selected for the cluster survey, to assess risk factors related to continued LF transmission and to pinpoint hotspots of transmission. One hundred houses were mapped in each village using GPS-enabled PDAs, and then 30 houses and 10 alternates were chosen for testing. All individuals in selected houses were asked to participate in a short survey about participation in MDA, history of residence in Leogane and general knowledge of LF. Survey teams returned to the houses at night to collect blood for antigen testing, microfilaremia and Bm14 antibody testing and collected mosquitoes from these communities in parallel. Antigen prevalence was highly variable among the 6 villages, with the highest being 38.2% (Dampus) and the lowest being 2.9% (Corail Lemaire); overall antigen prevalence was 18.5%. Initial cluster surveys of 2- to 4-year-old children were not related to community antigen prevalence. Nearest neighbor analysis found evidence of clustering of infection suggesting that LF infection was focal in distribution. Antigen prevalence among individuals who were systematically noncompliant with the MDAs, i.e. they had never participated, was significantly higher than among compliant individuals (p<0.05). A logistic regression model found that of the factors examined for association with infection, only noncompliance was significantly associated with infection. Thus, continuing transmission of LF seems to be linked to rates of systematic noncompliance.
Assuntos
Filariose Linfática/epidemiologia , Filariose Linfática/transmissão , Doenças Endêmicas , Filaricidas/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/sangue , Criança , Pré-Escolar , Análise por Conglomerados , Filariose Linfática/tratamento farmacológico , Feminino , Haiti/epidemiologia , Humanos , Masculino , Adesão à Medicação/estatística & dados numéricos , Pessoa de Meia-Idade , Fatores de Risco , Inquéritos e Questionários , Adulto JovemRESUMO
In addition to monitoring infection in the human host, there is also a need to assess larval infection in the vector mosquito population to evaluate the success of interventions for eliminating lymphatic filariasis transmission from endemic communities. Here, we review the current status of the available tools for quantifying vector infection and existing knowledge and evidence regarding potential infection thresholds for determining transmission interruption, to assess the potential for using vector infection monitoring as a tool for evaluating the success of filariasis treatment programmes.
Assuntos
Culicidae/parasitologia , Filariose Linfática/prevenção & controle , Filariose Linfática/transmissão , Controle de Mosquitos/métodos , Animais , Humanos , Insetos VetoresRESUMO
The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.
Assuntos
DNA de Helmintos/isolamento & purificação , DNA de Protozoário/isolamento & purificação , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reação em Cadeia da Polimerase/métodos , Wuchereria bancrofti/genética , Animais , Anopheles/parasitologia , Feminino , Humanos , Insetos Vetores/parasitologia , Papua Nova Guiné , Sensibilidade e Especificidade , Xenodiagnóstico/métodosRESUMO
Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund's adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia), compared with only 58% of vaccinated cattle (P = 0.015). This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate.
Assuntos
Antígenos de Helmintos/administração & dosagem , Bovinos , Modelos Animais de Doenças , Onchocerca/imunologia , Oncocercose/prevenção & controle , Vacinas/administração & dosagem , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Oncocercose/sangue , Oncocercose/imunologia , Oncocercose/parasitologia , Simuliidae , Vacinação , Vacinas/genética , Vacinas/imunologiaRESUMO
BACKGROUND: Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript. METHODOLOGY/PRINCIPAL FINDINGS: Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes. CONCLUSIONS/SIGNIFICANCE: This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites.
Assuntos
Brugia Malayi/fisiologia , Culicidae/parasitologia , Insetos Vetores/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Brugia Malayi/genética , DNA Complementar , DNA de Helmintos/genética , Larva/genética , Larva/fisiologiaRESUMO
BACKGROUND: This study employed various monitoring methods to assess the impact of repeated rounds of mass drug administration (MDA) on bancroftian filariasis in Papua New Guinea, which has the largest filariasis problem in the Pacific region. METHODOLOGY/PRINCIPAL FINDINGS: Residents of rural villages near Madang were studied prior to and one year after each of three rounds of MDA with diethylcarbamazine plus albendazole administered per World Health Organization (WHO) guidelines. The mean MDA compliance rate was 72.9%. Three rounds of MDA decreased microfilaremia rates (Mf, 1 ml night blood by filter) from 18.6% pre-MDA to 1.3% after the third MDA (a 94% decrease). Mf clearance rates in infected persons were 71%, 90.7%, and 98.1% after 1, 2, and 3 rounds of MDA. Rates of filarial antigenemia assessed by card test (a marker for adult worm infection) decreased from 47.5% to 17.1% (a 64% decrease) after 3 rounds of MDA. The filarial antibody rate (IgG(4) antibodies to Bm14, an indicator of filarial infection status and/or exposure to mosquito-borne infective larvae) decreased from 59.3% to 25.1% (a 54.6% decrease). Mf, antigen, and antibody rates decreased more rapidly in children <11 years of age (by 100%, 84.2%, and 76.8%, respectively) relative to older individuals, perhaps reflecting their lighter infections and shorter durations of exposure/infection prior to MDA. Incidence rates for microfilaremia, filarial antigenemia, and antifilarial antibodies also decreased significantly after MDA. Filarial DNA rates in Anopheles punctulatus mosquitoes that had recently taken a blood meal decreased from 15.1% to 1.0% (a 92.3% decrease). CONCLUSIONS/SIGNIFICANCE: MDA had dramatic effects on all filariasis parameters in the study area and also reduced incidence rates. Follow-up studies will be needed to determine whether residual infection rates in residents of these villages are sufficient to support sustained transmission by the An. punctulatus vector. Lymphatic filariasis elimination should be feasible in Papua New Guinea if MDA can be effectively delivered to endemic populations.
Assuntos
Albendazol/uso terapêutico , Dietilcarbamazina/uso terapêutico , Filariose Linfática/prevenção & controle , Adulto , Envelhecimento , Albendazol/administração & dosagem , Animais , Anopheles/parasitologia , Anti-Helmínticos/administração & dosagem , Anti-Helmínticos/uso terapêutico , Antígenos de Helmintos/sangue , Criança , Culicidae/parasitologia , DNA de Helmintos/genética , Dietilcarbamazina/administração & dosagem , Quimioterapia Combinada , Filariose Linfática/epidemiologia , Filariose Linfática/imunologia , Filariose/sangue , Filariose/epidemiologia , Filariose/imunologia , Humanos , Papua Nova Guiné/epidemiologia , Cooperação do Paciente , PrevalênciaRESUMO
Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.