Changes in Anti-OV-16 IgG4 Responses to Onchocerciasis after Elimination of Transmission in the Central Endemic Zone of Guatemala.

Current WHO guidelines for onchocerciasis elimination provide requirements for stopping mass drug administration of ivermectin and the verification of elimination of transmission. These guidelines also recommend post-elimination surveillance (PES) based on entomological surveys. Serological markers in humans could complement entomological PES once the longevity of anti-OV-16 antibody responses is better understood. In 2014-2015 we evaluated ELISA anti-OV-16 IgG4 antibody persistence among previously seropositive people from the central endemic zone of Guatemala. The country stopped all onchocerciasis program interventions in 2012 and was verified by WHO as having eliminated transmission of onchocerciasis in 2016. A total of 246 participants with prior OV-16 ELISA results from 2003, 2006, 2007, or 2009 were enrolled in a follow-up study. Of these, 77 people were previously OV-16 seropositive and 169 were previously seronegative. By 2014 and 2015, 56 (72.7%) previously seropositive individuals had sero-reverted, whereas all previous negatives remained seronegative. The progression of antibody responses over time was estimated using a mixed-effects linear regression model, using data from seropositive participants who had sero-reverted. The temporal variation showed a mean activity unit decay of 0.20 per year (95% credible interval [CrI]: 0.17, 0.23), corresponding to an estimated antibody response half-life of 3.3 years (95% CrI: 2.7, 4.1). These findings indicate that the majority of seropositive people will sero-revert over time.

Progress Toward Eradication of Dracunculiasis - Worldwide, January 2022-June 2023.

The effort to eradicate Dracunculus medinensis, the etiologic agent of dracunculiasis, or Guinea worm disease, commenced at CDC in 1980. In 1986, with an estimated 3.5 million cases worldwide in 20 African and Asian countries, the World Health Assembly called for dracunculiasis elimination. The Guinea Worm Eradication Program (GWEP) was established to help countries with endemic dracunculiasis reach this goal. GWEP is led by The Carter Center and supported by partners that include the World Health Organization, UNICEF, and CDC. In 2012, D. medinensis infections were unexpectedly confirmed in Chadian dogs, and since then, infections in dogs, cats, and baboons have posed a new challenge for GWEP, as have ongoing civil unrest and insecurity in some areas. By 2022, dracunculiasis was endemic in five countries (Angola, Chad, Ethiopia, Mali, and South Sudan), with only 13 human cases identified, the lowest yearly total ever reported. Animal infections, however, were not declining at the same rate: 686 animal infections were reported in 2022, including 606 (88%) in dogs in Chad. Despite these unanticipated challenges as well as the COVID-19 pandemic, countries appear close to reaching the eradication goal. GWEP will continue working with country programs to address animal infections, civil unrest, and insecurity, that challenge the eradication of Guinea worm.

Assembly and phylogeographical analysis of novel Taenia solium mitochondrial genomes suggest stratification within the African-American genotype.

BACKGROUND: Taenia solium is a parasite of public health concern, causing human taeniasis and cysticercosis. Two main genotypes have been identified: Asian and African-American. Although characterizing T. solium genotypes is crucial to understanding the genetic epidemiology of its diseases, not much is known about the differences between T. solium mitochondrial genomes from different genotypes. Also, little is known about whether genotypes are further subdivided. Therefore, this study aimed to identify a set of point mutations distributed throughout the T. solium mitochondrial genome that differentiate the African-American from the Asian genotype. Another objective was to identify whether T. solium main genotypes are further stratified. METHODS: One Mexican and two Peruvian T. solium mitochondrial genomes were assembled using reads available in the NCBI Sequence Read Archive and the reference genome from China as a template. Mutations with respect to the Chinese reference were identified by multiple genome alignment. Jensen-Shannon and Grantham scores were computed for mutations in protein-coding genes to evaluate whether they affected protein function. Phylogenies by Bayesian inference and haplotype networks were constructed using cytochrome c oxidase subunit 1 and cytochrome b from these genomes and other isolates to infer phylogeographical relationships. RESULTS: A set of 31 novel non-synonymous point mutations present in all genomes of the African-American genotype were identified. These mutations were distributed across the mitochondrial genome, differentiating the African-American from the Asian genotype. All occurred in non-conserved protein positions. Furthermore, the analysis suggested a stratification of the African-American genotypes into an East African and a West African sublineage. CONCLUSIONS: A novel set of 31 non-synonymous mutations differentiating the main T. solium genotypes was identified. None of these seem to be causing differences in mitochondrial protein function between parasites of the two genotypes. Furthermore, two sublineages within the African-American genotype are proposed for the first time. The presence of the East African sublineage in the Americas suggests an underestimated connection between East African and Latin American countries that might have arisen in the major slave trade between Portuguese Mozambique and the Americas. The results obtained here help to complete the molecular epidemiology of the parasite.

The limit of detection of the BioFire® FilmArray® gastrointestinal panel for the foodborne parasite Cyclospora cayetanensis.

Cyclosporiasis is a foodborne diarrheal illness caused by the parasite Cyclospora cayetanensis. The BioFire® FilmArray® gastrointestinal (FilmArray GI) panel is a common method for diagnosing cyclosporiasis from clinical stool samples. The currently published limit of detection (LOD) of this panel is in genome equivalents; however, it is unclear how this relates to the number of C. cayetanensis oocysts in a clinical sample. In this study, we developed a technique to determine the LOD in terms of oocysts, using a cell sorter to sort 1 to 50 C. cayetanensis oocyst(s) previously purified from three human stool sources. We found the FilmArray GI panel detected samples with ≥20 C. cayetanensis oocysts in 100% of replicates, with varying detection among samples with 1, 5, or 10 C. cayetanensis oocysts. This method provides a parasitologically relevant LOD that should enable comparison among C. cayetanensis detection techniques, including the FilmArray GI panel.

Retrospective evaluation of an integrated molecular-epidemiological approach to cyclosporiasis outbreak investigations - United States, 2021.

Cyclosporiasis results from an infection of the small intestine by Cyclospora parasites after ingestion of contaminated food or water, often leading to gastrointestinal distress. Recent developments in temporally linking genetically related Cyclospora isolates demonstrated effectiveness in supporting epidemiological investigations. We used 'temporal-genetic clusters' (TGCs) to investigate reported cyclosporiasis cases in the United States during the 2021 peak-period (1 May - 31 August 2021). Our approach split 655 genotyped isolates into 55 genetic clusters and 31 TGCs. We linked two large multi-state epidemiological clusters (Epidemiologic Cluster 1 [n = 136 cases, 54 genotyped] and Epidemiologic Cluster 2 [n = 42 cases, 15 genotyped]) to consumption of lettuce varieties; however, product traceback did not identify a specific product for either cluster due to the lack of detailed product information. To evaluate the utility of TGCs, we performed a retrospective case study comparing investigation outcomes of outbreaks first detected using epidemiological methods with those of the same outbreaks had TGCs been used to first detect them. Our study results indicate that adjustments to routine epidemiological approaches could link additional cases to epidemiological clusters of cyclosporiasis. Overall, we show that CDC's integrated genotyping and epidemiological investigations provide valuable insights into cyclosporiasis outbreaks in the United States.

Cyclospora cayetanensis comprises at least 3 species that cause human cyclosporiasis.

The apicomplexan parasite Cyclospora cayetanensis causes seasonal foodborne outbreaks of the gastrointestinal illness cyclosporiasis. Prior to the coronavirus disease-2019 pandemic, annually reported cases were increasing in the USA, leading the US Centers for Disease Control and Prevention to develop a genotyping tool to complement cyclosporiasis outbreak investigations. Thousands of US isolates and 1 from China (strain CHN_HEN01) were genotyped by Illumina amplicon sequencing, revealing 2 lineages (A and B). The allelic composition of isolates was examined at each locus. Two nuclear loci (CDS3 and 360i2) distinguished lineages A and B. CDS3 had 2 major alleles: 1 almost exclusive to lineage A and the other to lineage B. Six 360i2 alleles were observed ­ 2 exclusive to lineage A (alleles A1 and A2), 2 to lineage B (B1 and B2) and 1 (B4) was exclusive to CHN_HEN01 which shared allele B3 with lineage B. Examination of heterozygous genotypes revealed that mixtures of A- and B-type 360i2 alleles occurred rarely, suggesting a lack of gene flow between lineages. Phylogenetic analysis of loci from whole-genome shotgun sequences, mitochondrial and apicoplast genomes, revealed that CHN_HEN01 represents a distinct lineage (C). Retrospective examination of epidemiologic data revealed associations between lineage and the geographical distribution of US infections plus strong temporal associations. Given the multiple lines of evidence for speciation within human-infecting Cyclospora, we provide an updated taxonomic description of C. cayetanensis, and describe 2 novel species as aetiological agents of human cyclosporiasis: Cyclospora ashfordi sp. nov. and Cyclospora henanensis sp. nov. (Apicomplexa: Eimeriidae).

Progress Toward Global Eradication of Dracunculiasis - Worldwide, January 2021-June 2022.

Dracunculiasis (Guinea worm disease), caused by the parasite Dracunculus medinensis, is acquired by drinking water containing small crustacean copepods (water fleas) infected with D. medinensis larvae. Recent evidence suggests that the parasite also appears to be transmitted by eating fish or other aquatic animals. About 1 year after infection, the worm typically emerges through the skin on a lower limb of the host, causing pain and disability (1). No vaccine or medicine is available to prevent or treat dracunculiasis. Eradication relies on case containment* to prevent water contamination and other interventions to prevent infection, including health education, water filtration, treatment of unsafe water with temephos (an organophosphate larvicide), and provision of safe drinking water (1,2). CDC began worldwide eradication efforts in October 1980, and in 1984 was designated by the World Health Organization (WHO) as the technical monitor of the Dracunculiasis Eradication Program (1). In 1986, with an estimated 3.5 million cases† occurring annually in 20 African and Asian countries§ (3), the World Health Assembly called for dracunculiasis elimination. The Guinea Worm Eradication Program (GWEP),¶ led by The Carter Center and supported by partners that include WHO, UNICEF, and CDC, began assisting ministries of health in countries with endemic disease. In 2021, a total of 15 human cases were identified and three were identified during January-June 2022. As of November 2022, dracunculiasis remained endemic in five countries (Angola, Chad, Ethiopia, Mali, and South Sudan); cases reported in Cameroon were likely imported from Chad. Eradication efforts in these countries are challenged by infection in animals, the COVID-19 pandemic, civil unrest, and insecurity. Animal infections, mostly in domestic dogs, some domestic cats, and in Ethiopia, a few baboons, have now surpassed human cases, with 863 reported animal infections in 2021 and 296 during January-June 2022. During the COVID-19 pandemic all national GWEPs remained fully operational, implementing precautions to ensure safety of program staff members and community members. In addition, the progress toward eradication and effectiveness of interventions were reviewed at the 2021 and 2022 annual meetings of GWEP program managers, and the 2021 meeting of WHO's International Commission for the Certification of Dracunculiasis Eradication. With only 15 human cases identified in 2021 and three during January-June 2022, program efforts appear to be closer to reaching the goal of eradication. However, dog infections and impeded access because of civil unrest and insecurity in Mali and South Sudan continue to be the greatest challenges for the program. This report describes progress during January 2021-June 2022 and updates previous reports (2,4).

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.

Genotyping Cyclospora cayetanensis From Multiple Outbreak Clusters With An Emphasis on a Cluster Linked to Bagged Salad Mix-United States, 2020.

Cyclosporiasis is a diarrheal illness caused by the foodborne parasite Cyclospora cayetanensis. Annually reported cases have been increasing in the United States prompting development of genotyping tools to aid cluster detection. A recently developed Cyclospora genotyping system based on 8 genetic markers was applied to clinical samples collected during the cyclosporiasis peak period of 2020, facilitating assessment of its epidemiologic utility. While the system performed well and helped inform epidemiologic investigations, inclusion of additional markers to improve cluster detection was supported. Consequently, investigations have commenced to identify additional markers to enhance performance.

Inactivating Effects of Common Laboratory Disinfectants, Fixatives, and Temperatures on the Eggs of Soil Transmitted Helminths.

Soil-transmitted helminths (STH) are important and widespread intestinal pathogens of humans and animals. It is presently unknown which inactivating procedures may be universally effective for safe transport, preservation, and disinfection of STH-contaminated specimens, and this lack of knowledge may expose laboratory staff to higher risk of laboratory-acquired infections (LAI's). There are limited data on the efficacy of commonly used disinfectants and fecal fixatives for inactivating the eggs of STH. This work tested five disinfectants for surface cleanup, four storage temperature conditions, and six transport/storage fixatives, to inactivate eggs of three species of STH of animal origin (Ascaris suum "roundworm," Trichuris vulpis "whipworm" and Ancylostoma caninum "hookworm") as surrogates for human STH. Among disinfectants, exposure to 10% povidone-iodine for ≥5 min inactivated 100% of the three species tested, while 5 min exposure to 95% ethanol inactivated T. vulpis and A. caninum eggs. All of the fixatives tested had inactivation effects on A. caninum hookworm eggs within 24 h of exposure, except potassium dichromate, which required 48 h. 95% ethanol for ≥48 h inactivated eggs from all three STH species. Freezing at ≤-20°C for ≥24 h inactivated eggs of T. vulpis and A. caninum, but only freezing at -80°C for ≥24 h inactivated >99% eggs, including A. suum. This work provides an evidence base for health and safety guidelines and mitigation strategies for the handling, storage, and disposal of stool samples containing STH eggs in laboratory, health care, childcare, or veterinary settings. IMPORTANCE This study systematically evaluates common laboratory disinfectants and storage conditions for their effectiveness in inactivating the infective stages of soil-transmitted helminths (STH). Animal-infecting proxy species were chosen to represent three major groups of STH that infect humans: roundworms, whipworms, and hookworms. Previously published work in this area typically focuses on a particular inactivation method, either for a single STH species, or on a subset of closely related species. Because prediagnostic fecal specimens must be regarded as potentially infectious with a mix of species, such information may be of limited utility in a working laboratory. We provide a straightforward summary of storage and disinfection methods that can achieve complete inactivation across a range of STH species, which represents a significant advance for clinical, veterinary and research laboratory biosafety.

Progress Toward Global Eradication of Dracunculiasis, January 2020-June 2021.

Dracunculiasis (Guinea worm disease), caused by the parasite Dracunculus medinensis, is traditionally acquired by drinking water containing copepods (water fleas) infected with D. medinensis larvae, but in recent years also appears increasingly to be transmitted by eating fish or other aquatic animals. The worm typically emerges through the skin on a lower limb of the host 1 year after infection, causing pain and disability (1). There is no vaccine or medicine to prevent or medicine to treat dracunculiasis; eradication relies on case containment* to prevent water contamination and other interventions to prevent infection: health education, water filtration, treatment of unsafe water with temephos (an organophosphate larvicide), and provision of safe drinking water (1,2). The eradication campaign began in 1980 at CDC (1). In 1986, with an estimated 3.5 million cases† occurring annually in 20 African and Asian countries§ (3), the World Health Assembly called for dracunculiasis elimination (4). The Guinea Worm Eradication Program (GWEP), led by The Carter Center and supported by the World Health Organization (WHO), UNICEF, CDC, and other partners, began assisting ministries of health in countries with endemic disease. With 27 cases in humans reported in 2020, five during January-June 2021, and only six countries currently affected by dracunculiasis (Angola, Chad, Ethiopia, Mali, South Sudan, and importations into Cameroon), achievement of eradication appears to be close. However, dracunculiasis eradication is challenged by civil unrest, insecurity, and epidemiologic and zoologic concerns. Guinea worm infections in dogs were first reported in Chad in 2012. Animal infections have now overtaken human cases, with 1,601 reported animal infections in 2020 and 443 during January-June 2021. Currently, all national GWEPs remain fully operational, with precautions taken to ensure safety of program staff and community members in response to the COVID-19 pandemic. Because of COVID-19, The Carter Center convened the 2020 and 2021 annual GWEP Program Managers meetings virtually, and WHO's International Commission for the Certification of Dracunculiasis Eradication met virtually in October 2020. Since 1986, WHO has certified 199 countries, areas, and territories dracunculiasis-free. Six countries are still affected: five with endemic disease and importations into Cameroon. Seven countries (five with endemic dracunculiasis, Democratic Republic of the Congo, and Sudan) still lack certification (4). The existence of infected dogs, especially in Chad, and impeded access because of civil unrest and insecurity in Mali and South Sudan are now the greatest challenges to interrupting transmission. This report describes progress during January 2020-June 2021 and updates previous reports (2,4,5).

Investigation of US Cyclospora cayetanensis outbreaks in 2019 and evaluation of an improved Cyclospora genotyping system against 2019 cyclosporiasis outbreak clusters.

Cyclosporiasis is an illness characterised by watery diarrhoea caused by the food-borne parasite Cyclospora cayetanensis. The increase in annual US cyclosporiasis cases led public health agencies to develop genotyping tools that aid outbreak investigations. A team at the Centers for Disease Control and Prevention (CDC) developed a system based on deep amplicon sequencing and machine learning, for detecting genetically-related clusters of cyclosporiasis to aid epidemiologic investigations. An evaluation of this system during 2018 supported its robustness, indicating that it possessed sufficient utility to warrant further evaluation. However, the earliest version of CDC's system had some limitations from a bioinformatics standpoint. Namely, reliance on proprietary software, the inability to detect novel haplotypes and absence of a strategy to select an appropriate number of discrete genetic clusters would limit the system's future deployment potential. We recently introduced several improvements that address these limitations and the aim of this study was to reassess the system's performance to ensure that the changes introduced had no observable negative impacts. Comparison of epidemiologically-defined cyclosporiasis clusters from 2019 to analogous genetic clusters detected using CDC's improved system reaffirmed its excellent sensitivity (90%) and specificity (99%), and confirmed its high discriminatory power. This C. cayetanensis genotyping system is robust and with ongoing improvement will form the basis of a US-wide C. cayetanensis genotyping network for clinical specimens.

Surveillance of Human Guinea Worm in Chad, 2010-2018.

The total number of Guinea worm cases has been reduced by 99.9% since the mid-1980s when the eradication campaign began. Today, the greatest number of cases is reported from Chad. In this report, we use surveillance data collected by the Chad Guinea Worm Eradication Program to describe trends in human epidemiology. In total, 114 human cases were reported during the years 2010-2018, with highest rates of containment (i.e., water contamination prevented) in the years 2013, 2014, 2016, and 2017 (P < 0.0001). Approximately half of case-patients were female, and 65.8% of case-patients were aged 30 years or younger (mean: 26.4 years). About 34.2% of case-patients were farmers. Cases were distributed across many ethnicities, with a plurality of individuals being of the Sara Kaba ethnicity (21.3%). Most cases occurred between the end of June and the end of August and were clustered in the Chari Baguirmi (35.9%) and Moyen Chari regions (30.1%). Cases in the northern Chari River area peaked in April and in August, with no clear temporal pattern in the southern Chari River area. History of travel within Chad was reported in 7.0% of cases, and male case-patients (12.5%) were more likely than female case-patients (1.7%) to have reported a history of travel (P = 0.03). Our findings confirm that human Guinea worm is geographically disperse and rare. Although the proportion of case-patients with travel history is relatively small, this finding highlights the challenge of surveillance in mobile populations in the final stages of the global eradication campaign.

Cutaneous Leishmaniasis Caused by an Unknown Leishmania Strain, Arizona, USA.

We investigated an autochthonous case of cutaneous leishmaniasis caused by a genetically different Leishmania sp. in a patient in Arizona, USA. This parasite was classified into the subgenus Leishmania on the basis of multilocus DNA sequence and phylogenetic analyses of the rRNA locus and 11 reference genes.

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.

Development of a Multiplex Bead Assay for the Detection of Canine IgG4 Antibody Responses to Guinea Worm.

Increased levels of guinea worm (GW) disease transmission among dogs in villages along the Chari River in Chad threaten the gains made by the GW Eradication Program. Infected dogs with preemergent worm blisters are difficult to proactively identify. If these dogs are not contained, blisters can burst upon submersion in water, leading to the contamination of the water supply with L1 larvae. Guinea worm antigens previously identified using sera from human dracunculiasis patients were coupled to polystyrene beads for multiplex bead assay analysis of 41 non-endemic (presumed negative) dog sera and 39 sera from GW-positive dogs from Chad. Because commercially available anti-dog IgG secondary antibodies did not perform well in the multiplex assay, dog IgGs were partially purified, and a new anti-dog IgG monoclonal antibody was developed. Using the new 4E3D9 monoclonal secondary antibody, the thioredoxin-like protein 1-glutathione-S-transferase (GST), heat shock protein (HSP1)-GST, and HSP2-GST antigen multiplex assays had sensitivities of 69-74% and specificities of 73-83%. The domain of unknown function protein 148 (DUF148)-GST antigen multiplex assay had a sensitivity of 89.7% and a specificity of 85.4%. When testing samples collected within 1 year of GW emergence (n = 20), the DUF148-GST assay had a sensitivity of 90.0% and a specificity of 97.6% with a receiver-operating characteristic area under the curve of 0.94. Using sera from two experimentally infected dogs, antibodies to GW antigens were detected within 6 months of exposure. Our results suggest that, when used to analyze paired, longitudinal samples collected 1-2 months apart, the DUF148/GST multiplex assay could identify infected dogs 4-8 months before GW emergence.

Development of a Multiplex Bead Assay for the Detection of IgG Antibody Responses to Guinea Worm.

The success of the Guinea Worm (GW) Eradication Program over the past three decades has been tempered by the persistence of GW disease in a few African nations and the potential for a future resurgence in cases. Domestic dogs are now a major concern as a disease reservoir as large numbers of cases of canine GW disease are now reported each year, mainly along the Chari River in Chad. As a first step toward the development of a serologic assay for dogs, archived human plasma samples from dracunculiasis-positive donors from Togo were used to select adult female GW antigens for peptide sequencing and cloning. Eight protein sequences of interest were expressed as recombinant glutathione-S-transferase (GST) fusion proteins, and the most promising proteins were coupled to carboxylated microspheres for use in multiplex assays. A thioredoxin-like protein (TRXL1) and a domain of unknown function (DUF148) were assessed for total IgG and IgG4 reactivities using a panel of specimens from GW cases, uninfected donors, and individuals infected with various nematode worms, including Onchocerca volvulus. Both the DUF148-GST and the TRXL1-GST assays cross-reacted with O. volvulus sera, but the latter assay was always the more specific. The IgG4 and total IgG TRXL1-GST assays both had sensitivities > 87% and specificities > 90%. Maximum specificity (> 96%) was obtained with the total IgG assay when reactivity to both antigens was used to define a positive case. Given the good performance of the human assay, we are now working to modify the assay for dog assessments.

Duplex Real-Time PCR Assay for Clinical Differentiation of Onchocerca lupi and Onchocerca volvulus.

In the United States and Europe, human onchocerciasis is a rare disease caused by zoonotic or anthropophilic parasites in the genus Onchocerca. The zoonotic species identified in focal areas of Europe and United States is Onchocerca lupi, and Onchocerca volvulus, the anthroponotic species, may be found among people who had lived in endemic areas of Africa, the Arabian Peninsula, or Latin America. Onchocerciasis due to O. lupi is an emergent parasitic disease, with limited diagnostic methods, in addition to the lack of information on its biology, transmission, and epidemiology. Cutaneous nodules are the disease's most prevalent manifestation but lack diagnostic specificity. To address the diagnosis of onchocerciasis at reference laboratories, we developed a duplex TaqMan real-time PCR (qPCR) method, targeting the cytochrome oxidase subunit I locus which has species-specific probes to identify and differentiate O. lupi from O. volvulus. We determined the performance of the duplex with a panel of 45 samples: 11 positives for O. lupi, six for O. volvulus, five samples with negative results for Onchocerca spp., and 23 non-Onchocerca nematodes. The duplex qPCR correctly detected 10 of 11 O. lupi- and six of six O. volvulus-positive specimens. The new duplex assay allowed the simultaneous detection and discrimination of O. lupi and O. volvulus in clinical specimens, expediting and facilitating the clinical diagnosis of O. lupi in non-endemic settings where the disease is an infrequent finding.

Integrating Multiple Biomarkers to Increase Sensitivity for the Detection of Onchocerca volvulus Infection.

BACKGROUND: Serological assessments for human onchocerciasis are based on IgG4 reactivity against the OV-16 antigen, with sensitivities of 60-80%. We have previously identified 7 novel proteins that could improve serodiagnosis. METHODS: IgG4 responses to these 7 proteins were assessed by luciferase immunoprecipitation (LIPS) and enzyme-linked immunosorbent (ELISA) immunoassays. RESULTS: OVOC10469 and OVOC3261 were identified as the most promising candidates by IgG4-based immunoassays with sensitivities of 53% for rOVOC10469 and 78% for rOVOC3261 while specificity for each was >99%. These 2 antigens in combination with OV-16 increased the sensitivity for patent infections to 94%. The kinetics of appearance of these IgG4 responses based on experimentally infected non-human primates indicated that they were microfilarial- driven. Further, the IgG4 responses to both OVOC10469 and OVOC3261 (as well as to OV-16) drop significantly (p<0.05) following successful treatment for onchocerciasis. A prototype lateral flow rapid diagnostic test to detect IgG4 to both Ov-16 and OVOC3261 was developed and tested demonstrating an overall 94% sensitivity. CONCLUSION: The combined use of rOVOC3261 with OV-16 improved serologic assessment of O. volvulus infection, a current unmet need toward the goal of elimination of transmission of O. volvulus.

Cryptosporidium parvum as a risk factor of diarrhea occurrence in neonatal alpacas in Peru.

Cryptosporidiosis has been reported as an important cause of neonatal diarrhea and mortality in cattle, sheep, and other ruminants, but its impact on alpaca health has not been studied thoroughly. In this study, we have determined the prevalence and evaluated the role of cryptosporidiosis as a risk factor for diarrhea occurrence in newborn alpacas. During the calving season (January-March) of 2006, stool specimens (N = 1312) were collected from 24 herds of newborn alpacas in Puno and Cuzco, departments that account for the largest populations of alpacas in Peru. All the specimens were microscopically screened for Cryptosporidium spp. using the acid-fast technique. The association between Cryptosporidium detection and diarrhea was analyzed using χ2 test and generalized lineal model. Cryptosporidium species were determined by PCR-RFLP analysis of the small subunit rRNA gene. Cryptosporidium oocysts were detected in 159 of 1312 (12.4%) newborn alpacas. Results of the analyses demonstrated that crypstosporidiosis was significantly associated with diarrhea (PR = 3.84; CI95% 2.54-5.81; p < 0.0001). Only Cryptosporidium parvum was detected in the 153 Cryptosporidium-infected animals. Thus, there is an association of C. parvum infection with diarrhea in neonatal alpacas.