Uso de partículas magnéticas en la purificación de anticuerpos IgM contra Taenia solium / Use of magnetic particles in the purification of IgM antibodies against Taenia solium

RESUMEN Se evaluó el uso de partículas magnéticas acopladas a proteína L para la concentración y purificación de anticuerpos monoclonales inmunoglobulina M (mIgM) contra Taenia solium. Se evaluaron tres métodos de concentración y diferentes tiempos de elución y se optimizó la proporción de partículas a la proporción de mIgM. Demostramos que: 1) con el uso partículas magnéticas no se requiere de una concentración previa de mIgM, lo que disminuye la manipulación de los anticuerpos y mejora la recuperación, 2) se puede omitir el uso de un tampón de unión, ya que el pH de la mayoría de los sobrenadantes de cultivo celular son neutros, y 3) se necesitan tiempos de elución más largos (~45 minutos) para aumentar la recuperación a un nivel mayor a 80%. El estudio demuestra que el uso de partículas magnéticas acopladas a proteína L es una herramienta simple y eficiente para la concentración y purificación de mIgM.

ABSTRACT The use of L protein coupled magnetic particles for the concentration and purification of immunoglobulin M (mIgM) monoclonal antibodies against Taenia solium was evaluated. Three concentration methods and different elution times were evaluated and the ratio of particles to the ratio of mIgM was optimized. It is demonstrated that: 1) with the use of magnetic particles, a previous concentration of mIgM is not required, which reduces the manipulation of the antibodies and improves the recovery, 2) the use of a binding buffer can be omitted, since the pH of most cell culture supernatants are neutral, and 3) longer elution times (~ 45 minutes) are needed to increase recovery to a level greater than 80%. The study demonstrates that the use of L protein-coupled magnetic particles is a simple and efficient tool for mIgM concentration and purification.

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.

Identifying wildlife reservoirs of neglected taeniid tapeworms: Non-invasive diagnosis of endemic Taenia serialis infection in a wild primate population.

Despite the global distribution and public health consequences of Taenia tapeworms, the life cycles of taeniids infecting wildlife hosts remain largely undescribed. The larval stage of Taenia serialis commonly parasitizes rodents and lagomorphs, but has been reported in a wide range of hosts that includes geladas (Theropithecus gelada), primates endemic to Ethiopia. Geladas exhibit protuberant larval cysts indicative of advanced T. serialis infection that are associated with high mortality. However, non-protuberant larvae can develop in deep tissue or the abdominal cavity, leading to underestimates of prevalence based solely on observable cysts. We adapted a non-invasive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect circulating Taenia spp. antigen in dried gelada urine. Analysis revealed that this assay was highly accurate in detecting Taenia antigen, with 98.4% specificity, 98.5% sensitivity, and an area under the curve of 0.99. We used this assay to investigate the prevalence of T. serialis infection in a wild gelada population, finding that infection is substantially more widespread than the occurrence of visible T. serialis cysts (16.4% tested positive at least once, while only 6% of the same population exhibited cysts). We examined whether age or sex predicted T. serialis infection as indicated by external cysts and antigen presence. Contrary to the female-bias observed in many Taenia-host systems, we found no significant sex bias in either cyst presence or antigen presence. Age, on the other hand, predicted cyst presence (older individuals were more likely to show cysts) but not antigen presence. We interpret this finding to indicate that T. serialis may infect individuals early in life but only result in visible disease later in life. This is the first application of an antigen ELISA to the study of larval Taenia infection in wildlife, opening the doors to the identification and description of infection dynamics in reservoir populations.

Association between Taenia solium infection and HIV/AIDS in northern Tanzania: a matched cross sectional-study.

BACKGROUND: The frequency of Taenia solium, a zoonotic helminth, is increasing in many countries of sub-Saharan Africa, where the prevalence of the human immunodeficiency virus (HIV) is also high. However, little is known about how these two infections interact. The aim of this study was to compare the proportion of HIV positive (+) and negative (-) individuals who are infected with Taenia solium (TSOL) and who present with clinical and neurological manifestations of cysticercosis (CC). METHODS: In northern Tanzania, 170 HIV+ individuals and 170 HIV- controls matched for gender, age and village of origin were recruited. HIV staging and serological tests for TSOL antibodies (Ab) and antigen (Ag) were performed. Neurocysticercosis (NCC) was determined by computed tomography (CT) using standard diagnostic criteria. Neurological manifestations were confirmed by a standard neurological examination. In addition, demographic, clinical and neuroimaging data were collected. Further, CD4+ cell counts as well as information on highly active antiretroviral treatment (HAART) were noted. RESULTS: No significant differences between HIV+ and HIV- individuals regarding the sero-prevalence of taeniosis-Ab (0.6% vs 1.2%), CC-Ab (2.4% vs 2.4%) and CC-Ag (0.6% vs 0.0%) were detected. A total of six NCC cases (3 HIV+ and 3 HIV-) were detected in the group of matched participants. Two individuals (1 HIV+ and 1 HIV-) presented with headaches as the main symptom for NCC, and four with asymptomatic NCC. Among the HIV+ group, TSOL was not associated with CD4+ cell counts, HAART duration or HIV stage. CONCLUSIONS: This study found lower prevalence of taeniosis, CC and NCC than had been reported in the region to date. This low level of infection may have resulted in an inability to find cross-sectional associations between HIV status and TSOL infection or NCC. Larger sample sizes will be required in future studies conducted in that area to conclude if HIV influences the way NCC manifests itself.

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.

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.

Ring-screening to control endemic transmission of Taenia solium.

BACKGROUND: Taenia solium is a major cause of preventable epilepsy in developing nations. Screening and treatment of human intestinal stage infection (taeniasis) within high-risk foci may reduce transmission and prevent epilepsy by limiting human exposure to infective eggs. We piloted a ring-strategy that involves screening and treatment for taeniasis among households located nearby pigs heavily-infected with the larval stage (cysticercosis). These pigs mark areas of increased transmission and can be identified by tongue examination. METHODOLOGY: We selected two villages in northern Peru for a controlled prospective interventional cohort pilot study. In the intervention village (1,058 residents) we examined the tongues of all pigs every 4 months for nodules characteristic of cysticercosis. We then screened all residents living within 100-meters of any tongue-positive pig using enzyme-linked immunosorbent assay to detect Taenia antigens in stool. Residents with taeniasis were treated with niclosamide. In both the intervention and control (753 residents) we measured incidence of exposure by sampling the pig population every 4 months for serum antibodies against cysticercosis using enzyme-linked immunoelectrotransfer blot. PRINCIPAL FINDINGS: Baseline seroincidence among pigs born during the study was 22.6 cases per 100 pigs per-month (95% confidence interval [CI] 17.0-30.0) in the intervention and 18.1 (95% CI 12.7-25.9) in the control. After one year we observed a 41% reduction in seroincidence in the intervention village compared to baseline (incidence rate ratio 0.59, 95% CI 0.41-0.87) while the seroincidence in the control village remained unchanged. At study end, the prevalence of taeniasis was nearly 4 times lower in the intervention than in the control (prevalence ratio 0.28, 95% CI 0.08-0.91). CONCLUSIONS/SIGNIFICANCE: Ring-screening reduced transmission of T. solium in this pilot study and may provide an effective and practical approach for regions where resources are limited. However, this strategy requires validation in larger populations over a greater period of time.

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.

Neglected parasitic infections in the United States: cysticercosis.

Cysticercosis is a potentially fatal and preventable neglected parasitic infection caused by the larval form of Taenia solium. Patients with symptomatic disease usually have signs and symptoms of neurocysticercosis, which commonly manifest as seizures or increased intracranial pressure. Although there are many persons living in the United States who emigrated from highly disease-endemic countries and there are foci of autochthonous transmission of the parasite in the United States, little is known about burden and epidemiology of the disease in this country. In addition, despite advances in the diagnosis and management of neurocysticercosis, there remain many unanswered questions. Improving our understanding and management of neurocysticercosis in the United States will require improved surveillance or focused prospective studies in appropriate areas and allocation of resources towards answering some of the key questions discussed in this report.

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.

Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

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.

Geographic correlation between tapeworm carriers and heavily infected cysticercotic pigs.

BACKGROUND: Neurocysticercosis is a leading cause of preventable epilepsy in the developing world. Sustainable community-based interventions are urgently needed to control transmission of the causative parasite, Taenia solium. We examined the geospatial relationship between live pigs with visible cysticercotic cysts on their tongues and humans with adult intestinal tapeworm infection (taeniasis) in a rural village in northern Peru. The objective was to determine whether tongue-positive pigs could indicate high-risk geographic foci for taeniasis to guide targeted screening efforts. This approach could offer significant benefit compared to mass intervention. METHODS: We recorded geographic coordinates of all village houses, collected stool samples from all consenting villagers, and collected blood and examined tongues of all village pigs. Stool samples were processed by enzyme-linked immunosorbent assay (ELISA) for presence of Taenia sp. coproantigens indicative of active taeniasis; serum was processed by enzyme-linked immunoelectrotransfer blot for antibodies against T. solium cysticercosis (EITB LLGP) and T. solium taeniasis (EITB rES33). FINDINGS: Of 548 pigs, 256 (46.7%) were positive for antibodies against cysticercosis on EITB LLGP. Of 402 fecal samples, 6 (1.5%) were positive for the presence of Taenia sp. coproantigens. The proportion of coproantigen-positive individuals differed significantly between residents living within 100-meters of a tongue-positive pig (4/79, 5.1%) and residents living >100 meters from a tongue-positive pig (2/323, 0.6%) (p = 0.02). The prevalence of taeniasis was >8 times higher among residents living within 100 meters of a tongue-positive pig compared to residents living outside this range (adjusted PR 8.1, 95% CI 1.4-47.0). CONCLUSIONS: Tongue-positive pigs in endemic communities can indicate geospatial foci in which the risk for taeniasis is increased. Targeted screening or presumptive treatment for taeniasis within these high-risk foci may be an effective and practical control intervention for rural endemic areas.

Identification of antigenic targets for immunodetection of Balamuthia mandrillaris infection.

The free-living amoeba Balamuthia mandrillaris causes granulomatous amoebic encephalitis (GAE) in humans. Rapid identification of balamuthiasis is critical for effective therapeutic intervention and case management. In the present study we identified target antigens for the development of a serological assay for B. mandrillaris infection. We demonstrated by silver staining that protein profiles for all eight isolates of B. mandrillaris, independent of human or animal origin or geographic origin, appeared to be similar except for some minor differences, indicating the molecular homogeneity of these strains. The profiles of all isolates, which ranged from 200 to 10 kDa, were similar, with a prominent protein visible around 30 kDa; all appeared considerably different from protein profiles of the control E6 cells and Acanthamoeba castellanii and Naegleria fowleri isolates. Western blot analysis with rabbit hyperimmune serum identified the major immunodominant antigens of 25, 50, 75, and 80 kDa; positive human sera reacted strongly with proteins around 25, 40, 50, and 75 kDa. Proteins around 40 kDa detected by human serum were not recognized by hyperimmune rabbit serum. None of the target proteins were detected by uninfected control sera. Reactivities of five patients' sera with 4 different isolates of B. mandrillaris (2 strains of human and 2 strains of animal origins) revealed that patients' sera reacted slightly differently with different B. mandrillaris isolates, although major proteins of approximately 25, 50, and 75 kDa were present in all extracts.

A cross-sectional study of people with epilepsy and neurocysticercosis in Tanzania: clinical characteristics and diagnostic approaches.

Neurocysticercosis (NCC) is a major cause of epilepsy in regions where pigs are free-ranging and hygiene is poor. Pork production is expected to increase in the next decade in sub-Saharan Africa, hence NCC will likely become more prevalent. In this study, people with epilepsy (PWE, n=212) were followed up 28.6 months after diagnosis of epilepsy. CT scans were performed, and serum and cerebrospinal fluid (CSF) of selected PWE were analysed. We compared the demographic data, clinical characteristics, and associated risk factors of PWE with and without NCC. PWE with NCC (n=35) were more likely to be older at first seizure (24.3 vs. 16.3 years, p=0.097), consumed more pork (97.1% vs. 73.6%, p=0.001), and were more often a member of the Iraqw tribe (94.3% vs. 67.8%, p=0.005) than PWE without NCC (n=177). PWE and NCC who were compliant with anti-epileptic medications had a significantly higher reduction of seizures (98.6% vs. 89.2%, p=0.046). Other characteristics such as gender, seizure frequency, compliance, past medical history, close contact with pigs, use of latrines and family history of seizures did not differ significantly between the two groups. The number of NCC lesions and active NCC lesions were significantly associated with a positive antibody result. The electroimmunotransfer blot, developed by the Centers for Disease Control and Prevention, was more sensitive than a commercial western blot, especially in PWE and cerebral calcifications. This is the first study to systematically compare the clinical characteristics of PWE due to NCC or other causes and to explore the utility of two different antibody tests for diagnosis of NCC in sub-Saharan Africa.

Schistosomiasis among recreational users of Upper Nile River, Uganda, 2007.

After recreational exposure to river water in Uganda, 12 (17%) of 69 persons had evidence of schistosome infection. Eighteen percent self-medicated with praziquantel prophylaxis immediately after exposure, which was not appropriate. Travelers to schistosomiasis-endemic areas should consult a travel medicine physician.

Development and evaluation of a magnetic immunochromatographic test to detect Taenia solium, which causes taeniasis and neurocysticercosis in humans.

Taeniasis/cysticercosis caused by Taenia solium is a frequent parasitic infection of the human brain in most of the world. Rapid and simple screening tools to identify taeniasis and cysticercosis cases are needed for control programs, mostly to identify tapeworm carriers which are the source of infection and need to be treated, or as tools for point-of-care case detection or confirmation. These screening assays should be affordable, reliable, rapid, and easy to perform. Immunochromatographic tests meet these criteria. To demonstrate proof of principle, we developed and evaluated two magnetic immunochromatographic tests (MICTs) for detection of human Taenia solium taeniasis antibodies (ES33-MICT) and neurocysticercosis antibodies (T24-MICT). These assays detected stage-specific antibodies by using two recombinant proteins, rES33 for detection of taeniasis antibodies and rT24H for detection of cysticercosis antibodies. The sensitivity and specificity of the ES33-MICT to detect taeniasis infections were 94.5% and 96%, respectively, and those of the T24-MICT to detect cases of human cysticercosis with two or more viable brain cysts were 93.9% and 98.9%, respectively. These data provide proof of principle that the ES33- and T24-MICTs provide rapid and suitable methods to identify individuals with taeniasis and cysticercosis.

Multiantigen print immunoassay for comparison of diagnostic antigens for Taenia solium cysticercosis and taeniasis.

One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases.

Comparison of four selective media for the isolation of Burkholderia mallei and Burkholderia pseudomallei.

Currently there are no commercially available selective media indicated for the isolation of Burkholderia mallei and Burkholderia pseudomallei. Ashdown's agar, a custom selective medium for isolation of B. pseudomallei, is well described in the literature but unavailable commercially. Three commercially available media, Burkholderia cepacia selective agar (BCSA), oxidative-fermentative-polymyxin B-bacitracin-lactose (OFPBL) agar, and Pseudomonas cepacia (PC) agar are recommended for isolation of B. cepacia from respiratory secretions of cystic fibrosis patients. We evaluated the sensitivity and selectivity of these four media using 20 B. mallei, 20 B. pseudomallei, 20 Burkholderia spp., and 15 diagnostically challenging organisms. Ashdown's agar was the most sensitive medium for the isolation of B. pseudomallei, but it was unable to support growth of B. mallei. Pseudomonas cepacia agar was highly sensitive and selective for both organisms. In non-endemic areas, we suggest the use of the commercially available PC agar for the isolation of B. mallei and B. pseudomallei.