Development and validation of a quantitative PCR for the detection of Guinea worm (Dracunculus medinensis).

Dracunculus medinensis (Guinea worm) is a parasitic nematode that can cause the debilitating disease dracunculiasis (Guinea worm disease) in humans. The global Guinea Worm Eradication Program has led intervention and eradication efforts since the 1980s, and Guinea worm infections in people have decreased >99.99%. With the final goal of eradication drawing nearer, reports of animal infections from some remaining endemic countries pose unique challenges. Currently, confirmation of suspected Guinea worm infection relies on conventional molecular techniques such as polymerase chain reaction (PCR), which is not specific to Guinea worm and, therefore, requires sequencing of the PCR products to confirm the identity of suspect samples, a process that often takes a few weeks. To decrease the time required for species confirmation, we developed a quantitative PCR assay targeting the mitochondrial cytochrome b (cytb) gene of Guinea worm. Our assay has a limit of detection of 10 copies per reaction. The mean analytical parameters (± SE) were as follows: efficiency = 93.4 ± 7.7%, y-intercept = 40.93 ± 1.11, slope = -3.4896 ± 0.12, and the R2 = 0.999 ± 0.004. The assay did not amplify other nematodes found in Guinea worm-endemic regions and demonstrated 100% diagnostic sensitivity and specificity. Implementation of this quantitative PCR assay for Guinea worm identification could eliminate the need for DNA sequencing to confirm species. Thus, this approach can be implemented to provide more rapid confirmation of Guinea worm infections, leading to faster execution of Guinea worm interventions while increasing our understanding of infection patterns.

Epidemiological and molecular investigations of a point-source outbreak of Dracunculus medinensis infecting humans and dogs in Chad: a cross-sectional study.

BACKGROUND: Dracunculiasis (also known as Guinea worm disease), caused by the Dracunculus medinensis nematode, is progressing towards eradication, with a reduction in cases from 3·5 million cases in the mid-1980s to only 54 human cases at the end of 2019. Most cases now occur in Chad. On April 19, 2019, a 19-year-old woman presented with D medinensis in an area within the Salamat region of Chad, where the disease had not been previously reported. We aimed to investigate the connection between this case and others detected locally and elsewhere in Chad using a combination of epidemiological and genetic approaches. METHODS: In this cross-sectional field study, we conducted household case searches and informal group interviews in the Bogam, Liwi, and Tarh villages in Chad. All community members including children were eligible for participation in the outbreak investigation. Adult female D medinensis associated with this outbreak were collected for genetic analysis (18 from humans and two from dogs). Four mitochondrial genes and 22 nuclear microsatellite markers were used to assess relatedness of worms associated with the outbreak in comparison with other worms from elsewhere in Chad. FINDINGS: Between April 12 and Sept 6, 2019, we identified 22 human cases and two canine cases of dracunculiasis associated with 15 households. Six (40%) of the 15 affected households had multiple human or canine cases within the household. Most cases of dracunculiasis in people were from three villages in Salamat (21 [95%] of 22 cases), but one case was detected nearly 400 km away in Sarh city (outside the Salamat region). All people with dracunculiasis reported a history of consuming fish and unfiltered water. Worms associated with this outbreak were genetically similar and shared the same maternal lineage. INTERPRETATION: Molecular epidemiological results suggest a point-source outbreak that originated from a single female D medinensis, rather than newly identified sustained local transmission. The failure of the surveillance system to detect the suspected canine infection in 2018 highlights the challenge of canine D medinensis detection, particularly in areas under passive surveillance. Human movement can also contribute to dracunculiasis spread over long distances. FUNDING: The Carter Center.

Assessment of the Chad guinea worm surveillance information system: A pivotal foundation for eradication.

BACKGROUND: In the absence of a vaccine or pharmacological treatment, prevention and control of Guinea worm disease is dependent on timely identification and containment of cases to interrupt transmission. The Chad Guinea Worm Eradication Program (CGWEP) surveillance system detects and monitors Guinea worm disease in both humans and animals. Although Guinea worm cases in humans has declined, the discovery of canine infections in dogs in Chad has posed a significant challenge to eradication efforts. A foundational information system that supports the surveillance activities with modern data management practices is needed to support continued program efficacy. METHODS: We sought to assess the current CGWEP surveillance and information system to identify gaps and redundancies and propose system improvements. We reviewed documentation, consulted with subject matter experts and stakeholders, inventoried datasets to map data elements and information flow, and mapped data management processes. We used the Information Value Cycle (IVC) and Data-Information System-Context (DISC) frameworks to help understand the information generated and identify gaps. RESULTS: Findings from this study identified areas for improvement, including the need for consolidation of forms that capture the same demographic variables, which could be accomplished with an electronic data capture system. Further, the mental models (conceptual frameworks) IVC and DISC highlighted the need for more detailed, standardized workflows specifically related to information management. CONCLUSIONS: Based on these findings, we proposed a four-phased roadmap for centralizing data systems and transitioning to an electronic data capture system. These included: development of a data governance plan, transition to electronic data entry and centralized data storage, transition to a relational database, and cloud-based integration. The method and outcome of this assessment could be used by other neglected tropical disease programs looking to transition to modern electronic data capture systems.

Linked surveillance and genetic data uncovers programmatically relevant geographic scale of Guinea worm transmission in Chad.

BACKGROUND: Guinea worm (Dracunculus medinensis) was detected in Chad in 2010 after a supposed ten-year absence, posing a challenge to the global eradication effort. Initiation of a village-based surveillance system in 2012 revealed a substantial number of dogs infected with Guinea worm, raising questions about paratenic hosts and cross-species transmission. METHODOLOGY/PRINCIPAL FINDINGS: We coupled genomic and surveillance case data from 2012-2018 to investigate the modes of transmission between dog and human hosts and the geographic connectivity of worms. Eighty-six variants across four genes in the mitochondrial genome identified 41 genetically distinct worm genotypes. Spatiotemporal modeling revealed worms with the same genotype ('genetically identical') were within a median range of 18.6 kilometers of each other, but largely within approximately 50 kilometers. Genetically identical worms varied in their degree of spatial clustering, suggesting there may be different factors that favor or constrain transmission. Each worm was surrounded by five to ten genetically distinct worms within a 50 kilometer radius. As expected, we observed a change in the genetic similarity distribution between pairs of worms using variants across the complete mitochondrial genome in an independent population. CONCLUSIONS/SIGNIFICANCE: In the largest study linking genetic and surveillance data to date of Guinea worm cases in Chad, we show genetic identity and modeling can facilitate the understanding of local transmission. The co-occurrence of genetically non-identical worms in quantitatively identified transmission ranges highlights the necessity for genomic tools to link cases. The improved discrimination between pairs of worms from variants identified across the complete mitochondrial genome suggests that expanding the number of genomic markers could link cases at a finer scale. These results suggest that scaling up genomic surveillance for Guinea worm may provide additional value for programmatic decision-making critical for monitoring cases and intervention efficacy to achieve elimination.

Human infection with an unknown species of Dracunculus in Vietnam.

Guinea worm (GW) disease, caused by Dracunculus medinensis, is an almost eradicated waterborne zoonotic disease. The World Health Organization (WHO) currently lists GW as endemic in only five African countries. In July 2020, the Vietnamese public health surveillance system detected a hanging worm in a 23-year-old male patient, who did not report any travel to Africa or any country previously endemic for GW. The patient was hospitalized with symptoms of fatigue, anorexia, muscle aches, and abscesses, with worms hanging out of the skin in the lower limbs. The worms were retrieved from the lesions and microscopically examined in Vietnam, identifying structures compatible with Dracunculus spp. and L1-type larvae. A section of this parasite was sent to the Centers for Disease Control and Prevention (CDC) in Atlanta, United States, for confirmatory diagnosis of GW. The adult worm had cuticle structures compatible with Dracunculus parasites, although the length of L1 larvae was about 339 µm, substantially shorter than D. medinensis. DNA sequence analysis of the 18S small subunit rRNA gene confirmed that this parasite was not GW, and determined that the sample belonged to a Dracunculus sp. not previously reported in GenBank that clustered with the animal-infective Dracunculus insignis and Dracunculus lutrae, located in a different clade than D. medinensis. This study highlights the importance of effective public health surveillance systems and the collaborative work of local public health authorities from Vietnam with the WHO and CDC in efforts to achieve the eradication of GW.

Dracunculiasis in a domestic dog in Brazil.

We report and discuss the surprising encounter of a dog naturally infected by Dracunculus sp. in Brazil, a brief clinical history of the animal and a procedure for removing the nematode. We also present details on the morphology of the fragments collected from the nematode and a phylogenetic comparison of the partial sequences of the mitochondrial 18S rRNA and cytochrome c oxidase subunit I (COI) genes, deposited with others in GenBank. The samples were an independent lineage forming a well-supported monophyletic assemblage with D. medinensis. We thus conclude that this species has not yet been sequenced or even described and will only be elucidated by more information because only two species of Dracunculus have been reported in Brazil, D. fuelleborni and D. brasiliensis.

Population genomic evidence that human and animal infections in Africa come from the same populations of Dracunculus medinensis.

BACKGROUND: Guinea worm-Dracunculus medinensis-was historically one of the major parasites of humans and has been known since antiquity. Now, Guinea worm is on the brink of eradication, as efforts to interrupt transmission have reduced the annual burden of disease from millions of infections per year in the 1980s to only 54 human cases reported globally in 2019. Despite the enormous success of eradication efforts to date, one complication has arisen. Over the last few years, hundreds of dogs have been found infected with this previously apparently anthroponotic parasite, almost all in Chad. Moreover, the relative numbers of infections in humans and dogs suggests that dogs are currently the principal reservoir on infection and key to maintaining transmission in that country. PRINCIPAL FINDINGS: In an effort to shed light on this peculiar epidemiology of Guinea worm in Chad, we have sequenced and compared the genomes of worms from dog, human and other animal infections. Confirming previous work with other molecular markers, we show that all of these worms are D. medinensis, and that the same population of worms are causing both infections, can confirm the suspected transmission between host species and detect signs of a population bottleneck due to the eradication efforts. The diversity of worms in Chad appears to exclude the possibility that there were no, or very few, worms present in the country during a 10-year absence of reported cases. CONCLUSIONS: This work reinforces the importance of adequate surveillance of both human and dog populations in the Guinea worm eradication campaign and suggests that control programs aiming to interrupt disease transmission should stay aware of the possible emergence of unusual epidemiology as pathogens approach elimination.

Dracunculus Species in Meso-mammals from Georgia, United States, and Implications for the Guinea Worm Eradication Program in Chad, Africa.

The prevalence and diversity of parasitic nematodes in wildlife have been well studied for certain species, yet for others considerable gaps in knowledge exist. The parasitic nematode Dracunculus insignis infects North American wildlife, and past research on this species has led to an increased understanding of the potential host diversity and transmission of the closely related human Guinea worm, Dracunculus medinensis (which is currently the focus of a global eradication program). Many definitive hosts have been documented for D. insignis; however, the life cycle has been studied only in laboratories, and only a single phylogenetic study has been conducted on D. insignis (from Canada). The goals of the present study were to investigate the prevalence of infections with Dracunculus species among wildlife at a single site (Di-Lane plantation) in the southeastern United States, evaluate the genetic diversity of parasites at this site, and investigate potential paratenic hosts that may be involved in transmission. Over 3 yr, we sampled 228 meso-mammals, reporting an overall prevalence of infection with Dracunculus insignis of 20% (46/228). Amphibians and fish were sampled in the same geographic area as infected meso-mammals. Dracunculus insignis third-stage larvae were recovered from 2 different species of amphibians, but all fish sampled were negative. Phylogenetic analysis of the partial cytochrome c oxidase I (COI) gene showed very little diversity of Dracunculus at Di-Lane; however, we did recover a single nematode from a Virginia opossum (Didelphis virginiana) that falls outside of the D. insignis clade, more closely aligns with Dracunculus lutrae, and may represent an undescribed species. This work documents the occurrence of D. insignis in frogs, a potential transmission pathway for D. insignis at a single geographic site in nature. When applied to the global Guinea Worm Eradication Program, and Chad, Africa, in particular, this work increases our knowledge of the potential role of aquatic animals in the transmission of Dracunculus species and informs on potential intervention strategies that may be applied to the eradication of Guinea worm in Africa.

A pond-side test for Guinea worm: Development of a loop-mediated isothermal amplification (LAMP) assay for detection of Dracunculus medinensis.

Guinea worm Dracunculus medinensis causes debilitating disease in people and is subject to an ongoing global eradication programme. Research and controls are constrained by a lack of diagnostic tools. We developed a specific and sensitive LAMP method for detecting D. medinensis larval DNA in copepod vectors. We were able to detect a single larva in a background of field-collected copepods. This method could form the basis of a "pond-side test" for detecting potential sources of Guinea worm infection in the environment, in copepods, including in the guts of fish as potential transport hosts, enabling research, surveillance and targeting of control measures. The key constraint on the utility of this assay as a field diagnostic, is a lack of knowledge of variation in the temporal and spatial distribution of D. medinensis larvae in copepods in water bodies in the affected areas and how best to sample copepods to obtain a reliable diagnostic sample. These fundamental knowledge gaps could readily be addressed with field collections of samples across areas experiencing a range of worm infection frequencies, coupled with field and laboratory analyses using LAMP and PCR.

Guinea worm in domestic dogs in Chad: A description and analysis of surveillance data.

After a ten-year absence of reported Guinea worm disease in Chad, human cases were rediscovered in 2010, and canine cases were first recorded in 2012. In response, active surveillance for Guinea worm in both humans and animals was re-initiated in 2012. As of 2018, the Chad Guinea Worm Eradication Program (CGWEP) maintains an extensive surveillance system that operates in 1,895 villages, and collects information about worms, hosts (animals and humans), and animal owners. This report describes in detail the CGWEP surveillance system and explores epidemiological trends in canine Guinea worm cases during 2015-2018. Our results showed an increased in the number of canine cases detected by the system during the period of interest. The proportion of worms that were contained (i.e., water contamination was prevented) improved significantly over time, from 72.8% in 2015 to 85.7% in 2018 (Mantel-Haenszel chi-square = 253.3, P < 0.0001). Additionally, approximately 5% of owners of infected dogs reported that the dog had a Guinea worm-like infection earlier that year; 12.6% had a similar worm in a previous year. The proportion of dogs with a history of infection in a previous year increased over time (Mantel-Haenszel chi-square = 18.8, P < 0.0001). Canine cases were clustered in space and time: most infected dogs (80%) were from the Chari Baguirmi (38.1%) and Moyen Chari Regions (41.9%), and for each year the peak month of identified canine cases was June, with 78.5% occurring during March through August. Findings from this report evoke additional questions about why some dogs are repeatedly infected. Our results may help to target interventions and surveillance efforts in terms of space, time, and dogs susceptible to recurrent infection, with the ultimate goal of Guinea worm eradication.

Population genetic analysis of Chadian Guinea worms reveals that human and non-human hosts share common parasite populations.

Following almost 10 years of no reported cases, Guinea worm disease (GWD or dracunculiasis) reemerged in Chad in 2010 with peculiar epidemiological patterns and unprecedented prevalence of infection among non-human hosts, particularly domestic dogs. Since 2014, animal infections with Guinea worms have also been observed in the other three countries with endemic transmission (Ethiopia, Mali, and South Sudan), causing concern and generating interest in the parasites' true taxonomic identity and population genetics. We present the first extensive population genetic data for Guinea worm, investigating mitochondrial and microsatellite variation in adult female worms from both human and non-human hosts in the four endemic countries to elucidate the origins of Chad's current outbreak and possible host-specific differences between parasites. Genetic diversity of Chadian Guinea worms was considerably higher than that of the other three countries, even after controlling for sample size through rarefaction, and demographic analyses are consistent with a large, stable parasite population. Genealogical analyses eliminate the other three countries as possible sources of parasite reintroduction into Chad, and sequence divergence and distribution of genetic variation provide no evidence that parasites in human and non-human hosts are separate species or maintain isolated transmission cycles. Both among and within countries, geographic origin appears to have more influence on parasite population structure than host species. Guinea worm infection in non-human hosts has been occasionally reported throughout the history of the disease, particularly when elimination programs appear to be reaching their end goals. However, no previous reports have evaluated molecular support of the parasite species identity. Our data confirm that Guinea worms collected from non-human hosts in the remaining endemic countries of Africa are Dracunculus medinensis and that the same population of worms infects both humans and dogs in Chad. Our genetic data and the epidemiological evidence suggest that transmission in the Chadian context is currently being maintained by canine hosts.

Dracunculiasis Eradication: Are We There Yet?

This report summarizes the status of the global Dracunculiasis Eradication Program as of the end of 2017. Dracunculiasis (guinea worm disease) has been eliminated from 19 of 21 countries where it was endemic in 1986, when an estimated 3.5 million cases occurred worldwide. Only Chad and Ethiopia reported cases in humans, 15 each, in 2017. Infections of animals, mostly domestic dogs, with Dracunculus medinensis were reported in those two countries and also in Mali. Insecurity and infections in animals are the two main obstacles remaining to interrupting dracunculiasis transmission completely.

Prevalence, Structure, and Distribution of Novel Parasite Cysts Containing Dracunculus Species in River Otters ( Lontra canadensis) from Arkansas.

Adult female guinea worms ( Dracunculus spp.) usually are reported to occur singly in the extremities of their hosts, from which they deliver their larvae into the water through fistulae in the host's skin. We visually examined for the presence of worms in the fascia of the limbs of skinned carcasses of 184 river otters ( Lontra canadensis) harvested in Arkansas and report observations of cysts on wrists and ankles found on 12 otters. Cysts averaged 15.6 × 24.6 mm in diameter, were round to oval, and contained masses of up to 19 adult female Dracunculus sp. (mean 6.7). We speculate that high levels of infection in consumed paratenic hosts might have caused high infection rates, leading to large cyst formation in otters, as larvigerous Dracunculus sp. females accumulated in extremities. No males were discovered during the study, so identification of a sample of worms was based on molecular techniques.

Guinea Worm (Dracunculus medinensis) Infection in a Wild-Caught Frog, Chad.

A third-stage (infective) larva of Dracunculus medinensis, the causative agent of Guinea worm disease, was recovered from a wild-caught Phrynobatrachus francisci frog in Chad. Although green frogs (Lithobates clamitans) have been experimentally infected with D. medinensis worms, our findings prove that frogs can serve as natural paratenic hosts.

Renewed transmission of dracunculiasis--Chad, 2010.

Transmission of dracunculiasis (Guinea worm disease), a waterborne, parasitic disease targeted for eradication, was thought to have been interrupted in Chad since 2000, when the last case was reported. However, in 2010, 10 cases were confirmed by the Chad Ministry of Public Health (Ministère de la Santé Publique [MSP]) and the World Health Organization (WHO) during field investigations in which rumored cases were investigated and nearby villages were actively searched for additional cases. Because patients were not prevented from contaminating water sources, new cases were expected in 2011. During January-February 2011, MSP, WHO, and CDC conducted an investigation to gather additional information to guide prevention and response activities before the 2011 transmission season. Seven districts where cases had been confirmed or suspected in 2010 or where dracunculiasis was endemic during 1994-2000 were surveyed. The results of those surveys indicated that residents of 116 (55%) of 210 villages and 13 (87%) of 15 nomad camps consumed water from unsafe sources; 157 (75%) of 209 village key informants (KIs) and five (33%) of 15 nomad camp KIs knew about dracunculiasis. Thirty-one villages had confirmed or suspected cases during 2009-2011 and were classified as at-risk, requiring weekly active surveillance and urgent pre-positioning of materials for the 2011 transmission season. Nomadic populations are at risk for dracunculiasis because of unsafe water consumption and minimal knowledge of the disease. These populations also require targeted surveillance and prevention efforts (e.g., filter distribution, education, and case containment) to interrupt dracunculiasis transmission .

Differentiating Dracunculus medinensis from D. insignis, by the sequence analysis of the 18S rRNA gene.

This study, undertaken as a component of the global Dracunculiasis Eradication Program (DEP), was designed to provide molecular tools to distinguish Dracunculus medinensis, the nematode causing human dracunculiasis, from other tissue-dwelling nematodes, including other Dracunculus species that infect humans and other animals. DNA was extracted from D. medinensis and from a closely related species that infects North American carnivores, D. insignis, so that the genes coding for the small-subunit ribosomal RNA (18S rRNA) of the parasites could be amplified, sequenced and compared. Sequences were obtained for 20 specimens of D. medinensis (from humans in Pakistan, Yemen and six African countries endemic for dracunculiasis) and three of D. insignis (from raccoons trapped in the state of Georgia in the southern U.S.A.). All of the D. medinensis 18S-rRNA sequences were found to be 1819 bases long and identical. The three D. insignis 18S-rRNA sequences were also found to be identical to each other but were 1821 bases long and differed from the D. medinensis 18S- rRNA sequence at eight positions (representing a difference of 0.44%). The 18S-rRNA coding region of a Guinea worm extracted from a dog in Ghana was indistinguishable from that of the D. medinensis isolates from human cases. These results provide the basis for the molecular differentiation of D. medinensis that will permit the DEP to determine, rapidly and accurately, whether a worm recovered from an area considered dracunculiasis-free is a specimen of D. medinensis or not.