Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Low sensitivity and frequent cross-reactions in commercially available antibody detection ELISA assays for Taenia solium cysticercosis.

OBJECTIVE: To evaluate the diagnostic performance of two commercially available ELISA kits, Novalisa® and Ridascreen® , for the detection of antibodies to Taenia solium, compared to serological diagnosis of neurocysticercosis (NCC) by LLGP-EITB (electro-immunotransfer blot assay using lentil-lectin purified glycoprotein antigens). METHODS: Archive serum samples from patients with viable NCC (n = 45) or resolved, calcified NCC (n = 45), as well as sera from patients with other cestode parasites (hymenolepiasis, n = 45 and cystic hydatid disease, n = 45), were evaluated for cysticercosis antibody detection using two ELISA kits, Novalisa® and Ridascreen® . All NCC samples had previously tested positive, and all samples from heterologous infections were negative on LLGP-EITB for cysticercosis. Positive rates were calculated by kit and sample group and compared between the two kits. RESULTS: Compared to LLGP-EITB, the sensitivity of both ELISA assays to detect specific antibodies in patients with viable NCC was low (44.4% and 22.2%), and for calcified NCC, it was only 6.7% and 4.5%. Sera from patients with cystic hydatid disease were highly cross-reactive in both ELISA assays (38/45, 84.4%; and 25/45, 55.6%). Sera from patients with hymenolepiasis cross-reacted in five cases in one of the assays (11.1%) and in only one sample with the second assay (2.2%). CONCLUSIONS: The performance of Novalisa® and Ridascreen® was poor. Antibody ELISA detection cannot be recommended for the diagnosis of neurocysticercosis.

Seroprevalence of Baylisascaris procyonis Infection among Humans, Santa Barbara County, California, USA, 2014-2016.

Baylisascaris procyonis (raccoon roundworm) infection is common in raccoons and can cause devastating pathology in other animals, including humans. Limited information is available on the frequency of asymptomatic human infection. We tested 150 adults from California, USA, for B. procyonis antibodies; 11 were seropositive, suggesting that subclinical infection does occur.

Cross-reactivity of the 31 kDa antigen of Angiostrongylus cantonensis - Dealing with the immunodiagnosis of meningoencephalitis.

The primary causative agent of eosinophilic meningoencephalitis (EoM) in endemic regions is the nematode Angiostrongylus cantonensis. The occurrence of EoM was previously restricted to countries in Southeast Asia and the Pacific Islands; however, more recently, it has been reported from other regions, including Brazil. The commonly used diagnosis is detection of specific antibody reactivity to the 31 kDa antigen, which is derived from female worm somatic extracts. Here we report the occurrence of cross-reactivity to this antigen in sera from other parasitic infections, especially those that may cause EoM, such as gnathostomiasis, toxocariasis, hydatidosis and strongyloidiasis. We also demonstrated that the cross-reactivity, in part, is dependent of the concentration of antigen used in Western blot assays. We discuss the importance of these findings on the interpretation of this test.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray.

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcß4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core ß-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis.

Transmission of Babesia microti Parasites by Solid Organ Transplantation.

Babesia microti, an intraerythrocytic parasite, is tickborne in nature. In contrast to transmission by blood transfusion, which has been well documented, transmission associated with solid organ transplantation has not been reported. We describe parasitologically confirmed cases of babesiosis diagnosed ≈8 weeks posttransplantation in 2 recipients of renal allografts from an organ donor who was multiply transfused on the day he died from traumatic injuries. The organ donor and recipients had no identified risk factors for tickborne infection. Antibodies against B. microti parasites were not detected by serologic testing of archived pretransplant specimens. However, 1 of the organ donor's blood donors was seropositive when tested postdonation and had risk factors for tick exposure. The organ donor probably served as a conduit of Babesia parasites from the seropositive blood donor to both kidney recipients. Babesiosis should be included in the differential diagnosis of unexplained fever and hemolytic anemia after blood transfusion or organ transplantation.

Baylisascaris procyonis Roundworm Seroprevalence among Wildlife Rehabilitators, United States and Canada, 2012-2015.

Baylisascaris procyonis roundworms can cause potentially fatal neural larva migrans in many species, including humans. However, the clinical spectrum of baylisascariasis is not completely understood. We tested 347 asymptomatic adult wildlife rehabilitators for B. procyonis antibodies; 24 were positive, suggesting that subclinical baylisascariasis is occurring among this population.

Experimental transfusion-induced Babesia microti infection: dynamics of parasitemia and immune responses in a rhesus macaque model.

BACKGROUND: Babesiosis is an emerging tick-borne infection in humans. The increasing numbers of reported cases of transfusion-associated babesiosis (TAB), primarily caused by Babesia microti, represents a concern for the safety of the US blood supply. STUDY DESIGN AND METHODS: This study investigated kinetics of parasitemia and innate immune responses and dynamics of antibody responses during B. microti infection in rhesus macaques (RMs) using blood smears, quantitative polymerase chain reaction (qPCR), flow cytometry, and indirect fluorescent antibody testing. A total of six monkeys were transfused with either hamster or monkey-passaged B. microti-infected red blood cells (two and four monkeys, respectively) simulating TAB. RESULTS: The prepatent period in monkeys inoculated with hamster-passaged B. microti was 35 days compared with 4 days in monkeys transfused with monkey-passaged B. microti; the latter monkeys also had markedly higher parasitemia levels. The duration of the window period from the first detected parasitemia by qPCR analysis to the first detected antibody response ranged from 10 to 17 days. Antibody responses fluctuated during the course of the infection. Innate responses assessed by the frequencies of monocytes and activated B cells correlated with the kinetics and magnitude of parasitemia. On Day 14, additional activation peaks were noted for CD14+CD16+ and CD14-CD16+ monocytes and for CD11c+ myeloid dendritic cells, but only in animals transfused with monkey-passaged B. microti. Parasitemia persisted in these immunocompetent animals, similar to human infection. CONCLUSION: The results suggest that transfusion-associated transmission of B. microti leads to rapid onset of parasitemia (Day 4) in RMs, detectable antibody response 14 days later, and persistent parasitemia.

Taeniasis among Refugees Living on Thailand-Myanmar Border, 2012.

We tested refugee camp residents on the Thailand-Myanmar border for Taenia solium infection. Taeniasis prevalence was consistent with that for other disease-endemic regions, but seropositivity indicating T. solium taeniasis was rare. Seropositivity indicating cysticercosis was 5.5% in humans, and 3.2% in pigs. Corralling pigs and providing latrines may control transmission of these tapeworms within this camp.

Identification of immunodominant antigens for the laboratory diagnosis of toxocariasis.

OBJECTIVES: To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. METHODS: Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. RESULTS: Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. CONCLUSIONS: Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens.

Cysticercosis and epilepsy in rural Tanzania: a community-based case-control and imaging study.

OBJECTIVE: To assess the contribution of neurocysticercosis (NCC) to the burden of epilepsy in a rural Tanzanian population. METHODS: We identified adult people with epilepsy (PWE) in a door-to-door study in an established demographic surveillance site. PWE and community controls were tested for antibodies to Taenia solium, the causative agent of NCC, and all PWE were offered a computed tomography (CT) head scan. Data on household occupancy and sanitation, pig-keeping and pork consumption were collected from PWE and controls and associations with epilepsy were assessed using chi-square or Fisher's exact tests. RESULTS: Six of 218 PWE had antibodies to T. solium (2.8%; 95% CI 0.6-4.9), compared to none of 174 controls (Fisher's exact test, P = 0.04). Lesions compatible with NCC were seen in eight of 200 CT scans (4.0%; 95% CI 1.3-6.7). A total of 176 PWE had both investigations of whom two had positive serology along with NCC-compatible lesions on CT (1.1%; 95% 0.3-4.0). No associations between epilepsy and any risk factors for NCC were identified. CONCLUSIONS: Neurocysticercosis is present in this population but at a lower prevalence than elsewhere in Tanzania and sub-Saharan Africa. Insights from low-prevalence areas may inform public health interventions designed to reduce the burden of preventable epilepsy.

Recombinant protein- and synthetic peptide-based immunoblot test for diagnosis of neurocysticercosis.

One of the most well-characterized tests for diagnosing neurocysticercosis (NCC) is the enzyme-linked immunoelectrotransfer blot (EITB) assay developed at the CDC, which uses lentil lectin-bound glycoproteins (LLGP) extracted from Taenia solium cysticerci. Although the test is very reliable, the purification process for the LLGP antigens has been difficult to transfer to other laboratories because of the need for expensive equipment and technical expertise. To develop a simpler assay, we previously purified and cloned the diagnostic glycoproteins in the LLGP fraction. In this study, we evaluated three representative recombinant or synthetic antigens from the LLGP fraction, individually and in different combinations, using an immunoblot assay (recombinant EITB). Using a panel of 249 confirmed NCC-positive and 401 negative blood serum samples, the sensitivity of the recombinant EITB assay was determined to be 99% and the specificity was 99% for diagnosing NCC. We also tested a panel of 239 confirmed NCC-positive serum samples in Lima, Peru, and found similar results. Overall, our data show that the performance characteristics of the recombinant EITB assay are comparable to those of the LLGP-EITB assay. This new recombinant- and synthetic antigen-based assay is sustainable and can be easily transferred to other laboratories in the United States and throughout the world.

Comparative evaluation of 11 commercialized rapid diagnostic tests for detecting Trypanosoma cruzi antibodies in serum banks in areas of endemicity and nonendemicity.

Chagas disease is one of the main public health issues in Latin America. Increasingly during the past few decades, Trypanosoma cruzi infection has been detected in North America, Europe, and the Western Pacific, mainly as a result of population movement. The limited availability of rapid serological diagnostic tests hinders rapid diagnosis and early treatment in areas of endemicity and nonendemicity. In collaboration with 11 national reference laboratories (NRLs) from different geographical areas, we evaluated the performances of commercialized serological rapid diagnostic tests (RDT) for T. cruzi infection. Eleven commercialized T. cruzi infection RDTs were evaluated on a total of 474 samples extensively tested with at least three different techniques for Chagas disease, maintained at controlled low temperatures, and stored in the serum banks of the 11 NRLs. We measured the sensitivity, specificity, and concordance of each RDT and provided an additional questionnaire to evaluate its ease of use. The selected RDTs in this study were performed under controlled laboratory conditions. Out of the 11 RDTs, we found 8 of them to be useful, with the cassette format favored over the strip. We did not observe significant differences in RDT performances in the different regions. Overall, the performance results were lower than those disclosed by the manufacturers. The results of this evaluation validate the possibility of using RDTs to diagnose Chagas disease, thereby decreasing the time to treatment at a primary health care facility for patients who are willing to be treated. Further studies should be conducted in the laboratory and in the field to confirm these data, expressly to evaluate reproducibility in resource-limited settings, or using whole blood in clinical settings in areas of endemicity and nonendemicity.

Neglected parasitic infections: what every family physician needs to know.

Neglected parasitic infections, including Chagas disease, toxocariasis, cysticercosis, and toxoplasmosis, affect millions of persons in the United States. Relatively few resources have been devoted to surveillance, prevention, and treatment of these diseases. Chagas disease primarily affects Latin American immigrants and can cause heart failure and death if not treated. Immediate antiparasitic treatment is indicated for most patients with acute Chagas disease. Treatment is recommended for patients younger than 18 years who have chronic Chagas disease and is generally recommended for adults younger than 50 years who do not have advanced cardiomyopathy; treatment decisions for other patients should be made on an individual basis. Toxocariasis primarily affects children and can cause gastrointestinal, respiratory, and ophthalmologic disease. Treatment options include albendazole and mebendazole. Patients with ocular infection require referral to an ophthalmologist. Neurocysticercosis, a form of cysticercosis, is the most common infectious cause of seizures in some parts of the United States. Initial treatment should focus on symptom control. Humans generally acquire toxoplasmosis by eating undercooked contaminated meat or ingesting things that have been contaminated with cat feces. Congenital infection can result in miscarriage or adverse fetal effects. Treatment is recommended for immunosuppressed persons, pregnant women, and immunocompetent persons with severe symptoms.

High throughput sequencing of the Angiostrongylus cantonensis genome: a parasite spreading worldwide.

Angiostrongylus cantonensis is a parasitic nematode of rodents and a leading aetiological agent of eosinophilic meningitis in humans. Definitive diagnosis is difficult, often relying on immunodiagnostic methods which utilize crude antigens. New immunodiagnostic methods based on recombinant proteins are being developed, and ideally these methods would be made available worldwide. Identification of diagnostic targets, as well as studies on the biology of the parasite, are limited by a lack of molecular information on Angiostrongylus spp. available in databases. In this study we present data collected from DNA random high-throughput sequencing together with proteomic analyses and a cDNA walking methodology to identify and obtain the nucleotide or amino acid sequences of unknown immunoreactive proteins. 28 080 putative ORFs were obtained, of which 3371 had homology to other deposited protein sequences. Using the A. cantonensis genomic sequences, 156 putative ORFs, matching peptide sequences obtained from previous proteomic studies, were considered novel, with no homology to existing sequences. Full-length coding sequences of eight antigenic target proteins were obtained. In this study we generated not only the complete nucleotide sequences of the antigenic protein targets but also a large amount of genomic data which may help facilitate future genomic, proteomic, transcriptomic or metabolomic studies on Angiostrongylus.

Seroprevalence of antibodies against Taenia solium cysticerci among refugees resettled in United States.

Neurocysticercosis (NCC) is a disease caused by central nervous system infection by the larval stage of the pork tapeworm, Taenia solium. In developing countries, NCC is a leading cause of adult-onset epilepsy. Case reports of NCC are increasing among refugees resettled to the United States and other nations, but the underlying prevalence among refugee groups is unknown. We tested stored serum samples from the Centers for Disease Control and Prevention Migrant Serum Bank for antibodies against T. solium cysts by using the enzyme-linked immunoelectrotransfer blot. Seroprevalence was high among all 4 populations tested: refugees from Burma (23.2%), Lao People's Democratic Republic (18.3%), Bhutan (22.8%), and Burundi (25.8%). Clinicians caring for refugee populations should suspect NCC in patients with seizure, chronic headache, or unexplained neurologic manifestations. Improved understanding of the prevalence of epilepsy and other associated diseases among refugees could guide recommendations for their evaluation and treatment before, during, and after resettlement.

Workshop on research priorities for management and treatment of angiostrongyliasis(1).

In a concluding session of the workshop, the participants developed a list of 115 research and outreach needs, outlining the top 5-7 needs in each of 8 areas (Table). For complete information, including presenter details and abstracts, visit the workshop website at www.hawaii.edu/cowielab/Angio%20website%20home.htm.

The third described case of transfusion-transmitted Babesia duncani.

BACKGROUND: Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions. CASE REPORT: The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donor-a 67-year-old California resident-had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008. CONCLUSIONS: This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti.

Serologic surveillance of anthrax in the Serengeti ecosystem, Tanzania, 1996-2009.

Bacillus anthracis, the bacterium that causes anthrax, is responsible for varying death rates among animal species. Difficulties in case detection, hazardous or inaccessible carcasses, and misdiagnosis hinder surveillance. Using case reports and a new serologic assay that enables multispecies comparisons, we examined exposure to and illness caused by B. anthracis in different species in the Serengeti ecosystem in Tanzania during 1996-2009 and the utility of serosurveillance. High seroprevalence among carnivores suggested regular nonfatal exposure. Seropositive wildebeest and buffalo showed that infection was not invariably fatal among herbivores, whereas absence of seropositivity in zebras and frequent detection of fatal cases indicated high susceptibility. Exposure patterns in dogs reflected known patterns of endemicity and provided new information about anthrax in the ecosystem, which indicated the potential of dogs as indicator species. Serosurveillance is a valuable tool for monitoring and detecting anthrax and may shed light on mechanisms responsible for species-specific variability in exposure, susceptibility, and mortality rates.

Outbreak of leptospirosis among Adventure Race participants in Florida, 2005.

BACKGROUND: On 21 November 2005, a 32-year-old male resident of New York was hospitalized with suspected leptospirosis. He had participated in an endurance-length swamp race on 4-5 November 2005 outside of Tampa, Florida. METHODS: We interviewed racers to assess illness, medical care, and race activities. A suspected case was defined as fever plus > or = 2 signs or symptoms of leptospirosis occurring in a racer after 4 November 2005. Individuals with suspected cases were referred for treatment as needed and were asked to submit serum samples for microscopic agglutination testing (MAT) and for rapid testing by the dot enzyme-linked immunosorbent assay dipstick immunoglobulin M immunoassay. RESULTS: The Centers for Disease Control and Prevention and participating state health departments interviewed 192 (96%) of 200 racers from 32 states and Canada. Forty-four (23%) of 192 racers met the definition for a suspected case. The median age of the patients was 37 years (range, 19-66 years), and 128 (66.7%) were male. Fourteen (45%) of the 31 patients with suspected cases who were tested had their cases confirmed by serological testing (a single sample with MAT titer > or = 400), including the index case patient. Organisms of a potential novel serovar (species Leptospira noguchii) were isolated in culture from 1 case patient. Factors associated with increased risk of leptospirosis included swallowing river water (odds ratio [OR], 3.4; 95% confidence interval [CI], 1.6-7.0), swallowing swamp water (OR, 2.4; 95% CI, 1.1-5.2), and being submerged in any water (OR, 2.3; 95% CI, 1.1-4.7). CONCLUSIONS: This report describes a leptospirosis outbreak that resulted in a high rate of symptomatic infection among adventure racers in Florida. The growing popularity of adventure sports may put more people at risk for leptospirosis, even in areas that have not previously been considered areas of leptospirosis endemicity.