A rodent model for Dirofilaria immitis, canine heartworm: parasite growth, development, and drug sensitivity in NSG mice.

Heartworm disease, caused by Dirofilaria immitis, remains a significant threat to canines and felines. The development of parasites resistant to macrocyclic lactones (ML) has created a significant challenge to the control of the infection. The goal of this study was to determine if mice lacking a functional immune response would be susceptible to D. immitis. Immunodeficient NSG mice were susceptible to the infection, sustaining parasites for at least 15 weeks, with infective third-stage larvae molting and developing into the late fourth-stage larvae. Proteomic analysis of host responses to the infection revealed a complex pattern of changes after infection, with at least some of the responses directed at reducing immune control mechanisms that remain in NSG mice. NSG mice were infected with isolates of D. immitis that were either susceptible or resistant to MLs, as a population. The susceptible isolate was killed by ivermectin whereas the resistant isolate had improved survivability, while both isolates were affected by moxidectin. It was concluded that D. immitis survives in NSG mice for at least 15 weeks. NSG mice provide an ideal model for monitoring host responses to the infection and for testing parasites in vivo for susceptibility to direct chemotherapeutic activity of new agents.

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.

Elimination of onchocerciasis in Africa by 2025: the need for a broad perspective.

BACKGROUND: In response to the recent publication "Is onchocerciasis elimination in Africa feasible by 2025: a perspective based on lessons learnt from the African control programmes" by Dadzie et al., it is important to clarify and highlight the positive and unequivocal research and operational contributions from the American experience towards the worldwide elimination of human onchocerciasis (river blindness). MAIN TEXT: The strategies of twice or more rounds of mass drug administration (MDA) of ivermectin per year, as well as the use of OV-16 serology have allowed four American countries to be verified by World Health Organization to have eliminated transmission of Onchocerca volvulus, the etiological agent. These advances were also implemented in Sudan and Uganda; currently, both are the only African countries where ivermectin MDA was safely stopped in several transmission zones. CONCLUSIONS: Programmatic treatment and evaluation approaches, pioneered in the Americas, are the most efficient among the existing tools for elimination, and their broader use could catalyze the successful elimination of this disease in Africa.

A search for tiny dragons (Dracunculus medinensis third-stage larvae) in aquatic animals in Chad, Africa.

Dracunculus medinensis, or human Guinea worm (GW), causes a painful and debilitating infection. The global Guinea Worm Eradication Program (GWEP) has successfully reduced human GW cases from 3.5 million in 21 countries in 1986 to only 30 cases in three remaining countries in 2017. Since 2012, an increase in GW infections in domestic dogs, cats and baboons has been reported. Because these infections have not followed classical GW epidemiological patterns resulting from water-borne transmission, it has been hypothesized that transmission occurs via a paratenic host. Thus, we investigated the potential of aquatic animals to serve as paratenic hosts for D. medinensis in Chad, Africa. During three rainy and two dry season trips we detected no GW larvae in 234 fish, two reptiles and two turtles; however, seven GW larvae were recovered from 4 (1.4%) of 276 adult frogs. These data suggest GW infections may occur from ingestion of frogs but the importance of this route is unknown. Additional studies are needed, especially for other possible routes (e.g., ingestion of fish intestines that were recently shown to be a risk). Significantly, 150 years after the life cycle of D. medinensis was described, our data highlights important gaps in the knowledge of GW ecology.

Economic impact of malaria-related hospitalizations in the United States, 2000-2014.

BACKGROUND: Despite its elimination in the early 1950s, about 1700 cases of malaria are reported in the US every year. Few studies have quantified the direct and indirect costs of imported malaria in the US. METHODS: Disparities in the mean and total hospital days, hospital charges, and hospital costs for malaria-related hospitalizations in the US by demographic, clinical, species, financial, geographic, and institutional characteristics were examined using the 2000-2014 Nationwide Inpatient Sample (NIS). Trends and potential predictors for length of stay and hospital charges and costs were identified using negative binomial regression and linear regression, respectively. RESULTS: From 2000 to 2014, 22,029 malaria cases resulted in 95,948 hospital days for malaria-related hospitalizations, $176,391,466 in total hospital costs, and $555,435,849 in total charges. Mean charges increased significantly over the study period. Males, Blacks, and patients aged 25-44years accounted for the highest direct and indirect costs. Older age and having severe malaria was associated with a longer length of stay. Older age, severe malaria, HIV infection, and longer lengths of stay were associated with higher charges and costs. CONCLUSIONS: Malaria resulted in substantial direct and indirect costs in the US. Primary and secondary prevention measures should be prioritized among high-risk groups to reduce the economic burden.

Development of Onchocerca volvulus in humanized NSG mice and detection of parasite biomarkers in urine and serum.

BACKGROUND: The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice. CONCLUSIONS/SIGNIFICANCE: The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus.

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.

Evaluation of an OV-16 IgG4 Enzyme-Linked Immunosorbent Assay in Humans and Its Application to Determine the Dynamics of Antibody Responses in a Non-Human Primate Model of Onchocerca volvulus Infection.

Onchocerciasis is a neglected parasitic disease targeted for elimination. Current World Health Organization guidelines for elimination include monitoring antibody responses to the recombinant Onchocerca volvulus antigen OV-16 in children to demonstrate the absence of transmission. We report the performance characteristics of a modified OV-16 enzyme-linked immunosorbent assay (ELISA) and describe anti-OV-16 responses in serum samples from laboratory-inoculated nonhuman primates (NHPs) in relation to microfilariae (mf) in skin snip biopsies. This OV-16 IgG4 ELISA had sensitivity and specificity of 88.2% and 99.7%, respectively, as determined by receiver operator characteristic analysis using a serum panel of 110 positive and 287 negative samples from people infected with other filariae or other parasitic infections. Anti-OV-16 responses in inoculated NHP (N = 9) were evaluated at quarterly intervals for IgM and the four IgG subclasses. Enzyme-linked immunosorbent assay results showed a well-defined IgG4 reactivity pattern and moderate IgG1 antibody responses. Meanwhile, the reactivity by IgG2, IgG3, or IgM did not show a clear pattern. Temporal evolution of IgG4 reactivity was evaluated through monthly testing, showing that NHPs developed anti-OV-16 IgG4 on average at 15 months postinoculation (range: 10-18 months). The average time to detectable mf was also 15 months (range: 11-25). The OV-16 ELISA used in this study was robust and allowed the detection of IgG4 responses, which were observed only among animals with detectable mf (N = 5), four of which showed declines in antibody responses once mf cleared. These findings also confirmed that the most informative antibody subclass responses to OV-16 are IgG4.

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.

The role of national committees in eliminating onchocerciasis.

National onchocerciasis elimination committees (NOECs) serve to help ministries of health complete the pathway to successful verification of elimination of onchocerciasis (river blindness), as outlined in the 2016 World Health Organization guidelines. These guidelines, however, only take effect when the country believes it has reached a point that elimination can be demonstrated, and do not address the preceding milestones. Therefore, NOECs can be of great help with guiding and tailoring earlier planning, programming and assessments to empower national programs to aggressively move toward their countries' elimination goals. In this article, we provide suggestions for organizing NOECs and examples of four such committees that have successfully operated in Africa and the Americas.

Methylprednisolone acetate induces, and Δ7-dafachronic acid suppresses, Strongyloides stercoralis hyperinfection in NSG mice.

Strongyloides stercoralis hyperinfection causes high mortality rates in humans, and, while hyperinfection can be induced by immunosuppressive glucocorticoids, the pathogenesis remains unknown. Since immunocompetent mice are resistant to infection with S. stercoralis, we hypothesized that NSG mice, which have a reduced innate immune response and lack adaptive immunity, would be susceptible to the infection and develop hyperinfection. Interestingly, despite the presence of large numbers of adult and first-stage larvae in S. stercoralis-infected NSG mice, no hyperinfection was observed even when the mice were treated with a monoclonal antibody to eliminate residual granulocyte activity. NSG mice were then infected with third-stage larvae and treated for 6 wk with methylprednisolone acetate (MPA), a synthetic glucocorticoid. MPA treatment of infected mice resulted in 50% mortality and caused a significant >10-fold increase in the number of parasitic female worms compared with infected untreated mice. In addition, autoinfective third-stage larvae, which initiate hyperinfection, were found in high numbers in MPA-treated, but not untreated, mice. Remarkably, treatment with Δ7-dafachronic acid, an agonist of the parasite nuclear receptor Ss-DAF-12, significantly reduced the worm burden in MPA-treated mice undergoing hyperinfection with S. stercoralis Overall, this study provides a useful mouse model for S. stercoralis autoinfection and suggests a therapeutic strategy for treating lethal hyperinfection.

Risk factors for severe malaria among hospitalized patients in the United States, 2000-2014.

BACKGROUND: Factors associated with the development of severe malaria have not been well described for cases occurring in the United States (US). METHODS: Severe malaria hospitalizations data from the 2000-2014 Nationwide Inpatient Sample were analyzed. Frequencies were reported by demographic, clinical, species, financial, geographic, and institutional characteristics, and trends and disparities were identified. Logistic regression models were used to identify potential predictors for severe disease among those with malaria. RESULTS: From 2000 to 2014, there were an estimated 4823 severe malaria cases, representing 21.9% of all malaria-related hospitalizations, including 182 severe malaria deaths. Severe malaria was most common among inpatients who were male, Black, aged 45-64 years, and hospitalized in the South Atlantic division of the US. Older age was associated with higher odds of severe malaria, cerebral malaria, ARDS, severe anemia, and renal failure. Males had higher odds of developing renal failure and jaundice, while females had higher odds of developing severe anemia. HIV infection was associated with increased odds of severe malaria, severe anemia, and renal failure. CONCLUSION: Primary and secondary prevention measures, such as pre-travel consultations, chemoprophylaxis, and early diagnosis and treatment, should be emphasized and improved among high-risk prospective travelers to malaria endemic countries.

Progress Toward Global Eradication of Dracunculiasis, January 2016-June 2017.

Dracunculiasis (Guinea worm disease) is caused by Dracunculus medinensis, a parasitic worm. Approximately 1 year after a person acquires infection from contaminated drinking water, the worm emerges through the skin, usually on a lower limb (1). Pain and secondary bacterial infection can cause temporary or permanent disability that disrupts work and schooling. The campaign to eradicate dracunculiasis worldwide began in 1980 at CDC. In 1986, the World Health Assembly called for dracunculiasis elimination,* and the global Guinea Worm Eradication Program, led by the Carter Center and supported by the World Health Organization (WHO), United Nations Children's Fund, CDC, and other partners, began assisting ministries of health in countries with endemic dracunculiasis. In 1986, an estimated 3.5 million cases occurred each year in 20 countries in Africa and Asia (2). Since then, although the goal of eradicating dracunculiasis has not been achieved, considerable progress has been made. Compared with the 1986 estimate, the annual number of reported cases in 2016 has declined by >99%, and cases are confined to three countries with endemic disease. This report updates published (3-4) and unpublished surveillance data reported by ministries of health and describes progress toward dracunculiasis eradication during January 2016-June 2017. In 2016, a total of 25 cases were reported from three countries (Chad [16], South Sudan [six], Ethiopia [three]), compared with 22 cases reported from the same three countries and Mali in 2015 (Table 1). The 14% increase in cases from 2015 to 2016 was offset by the 25% reduction in number of countries with indigenous cases. During the first 6 months of 2017, the overall number of cases declined to eight, all in Chad, from 10 cases in three countries (Chad [four], South Sudan [four] and Ethiopia [two]) during the same period of 2016. Continued active surveillance, aggressive detection, and appropriate management of cases are essential eradication program components; however, epidemiologic challenges, civil unrest, and insecurity pose potential barriers to eradication.

Translating Research into Reality: Elimination of Lymphatic Filariasis from Haiti.

Research provides the essential foundation of disease elimination programs, including the global program to eliminate lymphatic filariasis (GPELF). The development and validation of new diagnostic tools and intervention strategies, critical steps in the evolution of GPELF, required a global effort. Lymphatic filariasis research in Haiti involved many partners and was directly linked to the development of the national elimination program and to the success achieved to date. Ongoing research efforts involving many partners will continue to be important in resolving the challenges faced by the program today in its final efforts to achieve elimination.

Possible Role of Fish as Transport Hosts for Dracunculus spp. Larvae.

To inform Dracunculus medinensis (Guinea worm) eradication efforts, we evaluated the role of fish as transport hosts for Dracunculus worms. Ferrets fed fish that had ingested infected copepods became infected, highlighting the importance of recommendations to cook fish, bury entrails, and prevent dogs from consuming raw fish and entrails.

Recurrence of Guinea Worm Disease in Chad after a 10-Year Absence: Risk Factors for Human Cases Identified in 2010-2011.

A decade after reporting its last case of Guinea worm disease (GWD), a waterborne parasitic disease targeted for eradication, Chad reported 20 confirmed human cases from 17 villages-10 cases in 2010 and 10 cases in 2011. In 2012, the first GWD dog infections were diagnosed. We conducted a case-control study during April-May 2012 to identify human transmission risk factors and epidemiologic links. We recruited 19 cases and 45 controls matched by age, sex, time, and location of exposure based on the case patients' periods of infection 10-14 months earlier. Data were analyzed with simple conditional logistic regression models using Firth penalized likelihood methods. Unusually, GWD did not appear to be associated with household primary water sources. Instead, secondary water sources, used outside the village or other nonprimary sources used at home, were risk factors (matched odds ratio = 38.1, 95% confidence interval = 1.6-728.2). This study highlights the changing epidemiology of GWD in Chad-household primary water sources were not identified as risk factors and few epidemiologic links were identified between the handfuls of sporadic cases per year, a trend that continues. Since this investigation, annual dog infections have increased, far surpassing human cases. An aquatic paratenic host is a postulated mode of transmission for both dogs and humans, although fish could not be assessed in this case-control study due to their near-universal consumption. GWD's evolving nature in Chad underscores the continued need for interventions to prevent both waterborne and potential foodborne transmission until the true mechanism is established.