Enzyme-linked Immunoelectrotransfer Blot Research Articles

Multiantigen print immunoassay (MAPIA) for the diagnosis of neurocysticercosis: a single-center diagnostic optimization and accuracy study in Lima, Peru.

Neurocysticercosis (NCC) is the most common helminthic infection of the human central nervous system. The antibody detection assay of choice is the enzyme-linked immunoelectrotransfer blot assay using lentil-lectin purified parasite antigens (LLGP-EITB, Western blot), an immunoassay with exceptional performance in clinical samples. However, its use is mainly restricted to a few research laboratories because the assay is labor-intensive and requires sophisticated equipment, expertise, and large amounts of parasite material for preparation of reagents. We report a new immunoprint assay (MAPIA) that overcomes most of these barriers. We initially compared the performance of five different antigen combinations in a subset of defined samples in the MAPIA format. After selecting the best-performing assay format (a combination of rGP50 + rT24H + sTs14 antigens), 148 archived serum samples were tested, including 40 from individuals with parenchymal NCC, 40 with subarachnoid NCC, and 68 healthy controls with no evidence of neurologic disease. MAPIA using three antigens (rGP50 + rT24H + sTs14) was highly sensitive and specific for detecting antibodies in NCC. It detected 39 out of 40 (97.5%) parenchymal NCC cases and 40/40 (100%) subarachnoid cases and was negative in 67 out of 68 (98.53%) negative samples. MAPIA using three recombinant and synthetic antigens is a simple and economical tool with a performance equivalent to the LLGP-EITB assay for the detection of specific antibodies to NCC. The MAPIA overcomes existing barriers to adoption of the EITG LLGP and is a candidate for worldwide use.

Serological and molecular detection of neurocysticercosis among epileptic patients in Nagpur, Maharashtra state (India).

Neurocysticercosis (NCC), one of the most important neuroparasitic diseases in humans, is caused by Cysticercus cellulosae, the metacestode stage of digenetic zoonotic cestode Taenia solium. The present study aims at the detection of anti-cysticercus antibodies in the sera of epileptic patients (n=26) visiting a tertiary care hospital in Nagpur, Maharashtra state, India, by an in-house developed indirect IgG-ELISA and enzyme-linked immunoelectro transfer blot (EITB) assay using different antigens (namely, Whole Cyst Antigen (WCA), Cystic Fluid Antigen (CFA), Scolex Antigen (SA), Excretory-Secretory Antigen (ESA) and Membrane-Body Antigen (MBA)) prepared from T. solium metacestodes to find out the status of NCC. An attempt has also been made for molecular detection of NCC from blood samples of those patients by Polymerase Chain Reaction (PCR) assay targeted at large subunit rRNA gene of T. solium. The IgG ELISA level of anti-cysticercus antibodies against WCA, CFA, SA, ESA and MBA antigens were as follows: 19.23 %, 23.07 %, 38.46 %, 30.76 % and 15.38 %. The seroreactivity to CFA, SA and ESA was found in equal proportions in patients with ring-enhancing lesions. In the EITB assay, the lower and medium molecular weight protein bands of SA and ESA were immunodominant compared to the higher WCA and CFA peptides. PCR positivity could be observed in 34.6 % (9/26) of the patients under study. It is the first report of detecting NCC among epileptic patients of the Nagpur region of Maharashtra state in India using serological and molecular tools.

Triplex ELISA for Assessing Durability of Taenia solium Seropositivity after Neurocysticercosis Cure.

Neurocysticercosis prevalence estimates often are based on serosurveys. However, assessments of Taenia solium seropositivity durability in patients with various neurocysticercosis types are lacking. We optimized a triplex serologic ELISA by using synthetic GP50, T24H, and Ts18var3 antigens for T. solium. We used that assay to test sequential serologic responses over several years after neurocysticercosis cure in 46 patients, 9 each with parenchymal or ventricular neurocysticercosis and 28 with subarachnoid disease. Triplex results were concordant with 98% of positive and 100% of negative enzyme-linked immunoelectrotransfer blots. Eight years after neurocysticercosis cure, 11.1% of patients with parenchymal, 47.3% with subarachnoid, and 41.7% with ventricular disease were still seropositive. Median time to seroreversion after cure in this cohort in a T. solium nonendemic area was 2 years for parenchymal disease, 4 years for ventricular disease, and 8 years for subarachnoid disease. Our findings can inform epidemiologic models that rely on serosurveys to estimate disease burden.

Şap Hastalığı Teşhisi için Enzim Immunotransfer Blot Testinin kantitatif bir yaklaşım ile iyileştirilmesi

Foot-and-mouth disease (FMD) is a highly contagious animal disease that causes devastating economic losses. The trade of live animals and derived products is only possible if the exporting country is free from disease, according to the World Organization for Animal Health (WOAH) code for FMD. One of the most important ways to prove disease-free is to measure the levels of non-structural protein antibodies (NPS) of FMD virus in the target population sampled. For detection of the disease status of a herd, mass screening and assays such as Enzyme-linked immunosorbent assay (ELISA) and Enzyme-linked immunoelectrotransfer blot assay (EITB) were developed and described in the WOAH diagnostic manual. In this study, recombinant FMDV NS proteins were produced and tested with sera panels collected from uninfected and naturally infected animals using a quantitative Western blot assay as an improved EITB, which enables numerical documentation and statistical analysis. NSP band intensities were used to determine the cut-off values, differentiating infected from non-infected animals and revealing variable sensitivity among the different NSPs. The quantitative EITB results also showed a correlation with the NSP-ELISA results.

Improved Dot-ELISA Assay Using Purified Sheep Coenurus cerebralis Antigenic Fractions for the Diagnosis of Zoonotic Coenurosis

Clinicians face significant problems in the diagnosis of zoonotic coenurosis. The current study aimed to develop an improved dot-Enzyme-linked-immunosorbent assay (dot-ELISA) for the diagnosis of zoonotic coenurosis using sheep Coenurus cerebralis scolices purified antigen (CcS-Ag) and to compare the obtained results with those of indirect ELISA and Enzyme-linked immunoelectrotransfer blot technique (EITB). Sera were collected from humans and sheep infected or suspected of infection with Coenurus cerebralis, control cases, and cases infected with other parasites. The CcS-Ag was proved to be the most specific antigen. This antigen was fractionated, and its specific polypeptides against anti-C. cerebralis antibodies (ACc-Ab) were identified using EITB. Fractions at the molecular weight (MW) of 48 and 58 kDa were proved as the only specific ones, eluted from the gel and concentrated, then dotted on the NC sheet as pooled antigen before its evaluation in the diagnosis of infection using dot-ELISA. Dot-ELISA demonstrated absolute 100% sensitivity and 100% specificity as recorded by EITB, compared to both fractions on a nitrocellulose (NC) sheet using surgically proved infected human or sheep sera as a gold standard. Diagnosis by ELISA using crude CcS-Ag revealed similar sensitivity but lower specificity (75%). The diagnostic accuracy of dot-ELISA was proved by comparing its results with postmortem data obtained post slaughtering of 20 suspected sheep and patients investigated by computed tomography (CT) and magnetic resonance imaging (MRI). In conclusion, the selection of specific fractions after EITB to be used in dot-ELISA improved the diagnostic value of the test as a diagnostic tool gathering the benefits of ELISA and EITB.

Characterization of somatic and metabolic antigens of Cysticercus cellulosae and determination of immunodominant proteins

The present study shows the characterization of four somatic antigens (Whole Cyst Antigen (WCA), Cystic Fluid Antigen (CFA), Scolex Antigen (SA) and Membrane-Body Antigen (MBA)) and one metabolic antigen (Excretory-Secretory Antigen (ESA)) prepared from Cysticercus cellulosae originated from a naturally infected pig. Immunodominat proteins were determined using hyper immune rabbit sera. The Sodium Dodecyl SulphatePolyacrylamide Gel Electrophoresis (SDS-PAGE) profile of the antigens revealed different numbers and patterns of protein bands in the range of 11.80 to 176.74 kDa. The electrophoretic pattern of ESA also revealed a number of closely placed indistinct bands in the range of 20-34 kDa.. The proteins in the lower molecular weight range (< 30 kDa) in the case of WCA, CFA, ESA, and the medium molecular weight range (30-60 kDa) in the case of SA and MBA were observed to be immunodominant in the Enzyme-Linked Immunoelectrotransfer Blot (EITB) assay. These immunodominant proteins could be further investigated as potential diagnostic antigens in the serodiagnosis of cysticercosis in pigs, and neurocysticercosis in human patients.

Evaluation of recombinant glutathione transferase 26 kDa, thioredoxin-1, and endophilin B1 of Taenia solium in the diagnosis of human neurocysticercosis

Neurocysticercosis caused by Taenia solium larvae is a neglected disease that persists in several countries, including Mexico, and causes a high disability-adjusted life year burden. Neuroimaging tools such as computed tomography and magnetic resonance imaging are the most efficient for its detection, but low availability and high costs in most endemic regions limit their use. Serological methods such as lentil lectin-purified glycoprotein enzyme-linked immunoelectrotransfer blot antibody detection and monoclonal antibody-based enzyme-linked immunosorbent assays for HP10 antigen detection have been useful in supporting the diagnosis of this disease. We evaluated three T. solium recombinant antigens: glutathione transferase of 26 kDa (Ts26GST); thioredoxin-1 (TsTrx-1), and endophilin B1 (TsMEndoB1) by EITB. These are antigenic proteins antigenic, abundant in excretion/secretion products of the parasite, and do not cross-react with homologous host proteins. Ts26GST and TsTrx-1 showed sensitivity of 79 and 88%, specificity of 86 and 97%, PPV of 83 and 97% and NPV of 82 and 91%, respectively, for neurocysticercosis diagnosis. The recombinant antigens allowed the diagnosis of 70% (Ts26GST) and 80% (TsTrx-1) of patients having only one cysticercus. Further studies on specific regions of these proteins could improve T. solium diagnostics.

Improved Diagnosis of Viable Parenchymal Neurocysticercosis by Combining Antibody Banding Patterns on Enzyme-Linked Immunoelectrotransfer Blot (EITB) with Antigen Enzyme-Linked Immunosorbent Assay (ELISA).

The diagnosis of neurocysticercosis (NCC) depends on neuroimaging and serological confirmation. While antibody detection by enzyme-linked immunoelectrotransfer blot (EITB) fails to predict viable NCC, EITB banding patterns provide information about the host's infection course. Adding antigen enzyme-linked immunosorbent assay (Ag-ELISA) results to EITB banding patterns may improve their ability to predict or rule out of viable NCC. We assessed whether combining EITB banding patterns with Ag-ELISA improves discrimination of viable infection in imaging-confirmed parenchymal NCC. EITB banding patterns were grouped into classes using latent class analysis. True-positive and false-negative Ag-ELISA results in each class were compared using Fisher's exact test. Four classes were identified: 1, EITB negative or positive to GP50 alone (GP50 antigen family); 2, positive to GP42-39 and GP24 (T24/42 family), with or without GP50; and 3 and 4, positive to GP50, GP42-39, and GP24 and reacting to bands in the 8-kDa family. Most cases in classes 3 and 4 had viable NCC (82% and 88%, respectively) compared to classes 2 and 1 (53% and 5%, respectively). Adding positive Ag-ELISA results to class 2 predicted all viable NCC cases (22/22 [100%]), whereas 11/40 patients (27.5%) Ag-ELISA negative had viable NCC (P < 0.001). Only 1/4 patients (25%) Ag-ELISA positive in class 1 had viable NCC, whereas 1/36 patients (2.8%) Ag-ELISA negative had viable NCC (P = 0.192). In classes 3 and 4, adding Ag-ELISA was not contributory. Combining Ag-ELISA with EITB banding patterns improves discrimination of viable from nonviable NCC, particularly for class 2 responses. Together, these complement neuroimaging more appropriately for the diagnosis of viable NCC.

Evaluation of an Antibody Detecting Point of Care Test for Diagnosis of Taenia solium Cysticercosis in a Zambian Rural Community: A Prospective Diagnostic Accuracy Study.

The lack of cheap, easy-to-use, rapid diagnostic tests has led to the development of several rapid diagnostic tests for cysticercosis. The new prototype two-strip, Taenia solium point of care test (TS POC) detects antibodies against taeniosis (TS POC T) and cysticercosis (TS POC CC). This study evaluated the diagnostic performance of the TS POC CC in the Sinda district in eastern Zambia. A sample of 1254 participants was recruited and tested with the TS POC. Out of the 1249 participants with a valid TS POC result, 177 (14%) tested positive while 1072 (86%) tested negative. All individuals with a positive TS POC and a subset of negative TS POC participants were selected for serum sampling, and were subjected to the recombinant glycoprotein T24H enzyme-linked immunoelectrotransfer blot (rT24H EITB) and the serum B60/158 (serum Ag) enzyme-linked immunosorbent assay (Ag ELISA). Performance characteristics were estimated using a Bayesian approach with probabilistic constraints. Based on 255 complete cases, the estimated sensitivity and specificity of the TS POC CC test were 35% (95% CI: 14–63%) and 87% (95% CI: 83–90%), respectively. The diagnostic performance needs to be improved, possibly by titrating antigen and other reagents’ concentration in the strip to produce a performance similar to existing cysticercosis tests such as the rT24H EITB.

Challenges Encountered When Evaluating an Antibody-Detecting Point-of-Care Test for Taeniosis in an Endemic Community in Zambia: A Prospective Diagnostic Accuracy Study.

Taenia solium taeniosis diagnosis is challenging because current tests perform sub-optimally and/or are expensive, require sophisticated equipment, infrastructure and trained manpower, and therefore are not community deployable. A recently-developed, multi-strip, T. solium point-of-care test (TS POC) for simultaneous detection of tapeworm (TS POC T) and cysticercus (TS POC CC) human antibodies was evaluated for diagnostic accuracy on consecutively recruited community participants in Sinda district, Zambia. All participants were tested using the TS POC test. All test-positives and 20% of the test-negative participants were invited to give a blood and stool sample for reference testing. Three different reference tests were used for taeniosis diagnosis: recombinant rES33 enzyme-linked immunoelectrotransfer blot (rES33 EITB), copro PCR and copro Ag ELISA. Bayesian analysis with probabilistic constraints was used to estimate sensitivity and specificity. In total, 1254 participants were tested with the TS POC test, of whom 13 tested positive using the TS POC T. Based on 161 participants with complete data, the estimated sensitivity and specificity for the TS POC T test were 38% (95% CI: 5–93%) and 99% (95% CI: 98–100%), respectively. The challenge of highly variable inter-assay performance is highlighted. We recommend either increasing the sensitivity or redesigning the test.

Imaging correlates of serum enzyme-linked immunoelectrotransfer blot (EITB) positivity in patients with parenchymal neurocysticercosis: results from 521 patients.

The presence of perilesional edema among patients with parenchymal neurocysticercosis (pNCC) of various lesion subtypes has not been correlated with results of serum enzyme-linked immunotransfer blot (EITB) for cysticercal antibodies. In total, 521 patients with pNCC were classified into solitary cysticercus granuloma (SCG), multiple lesions, at least one of which was an enhancing granuloma (GMNCC), solitary calcified cysticercal lesion (SCC) and multiple calcified cysticercal lesions (CMNCC). The proportion of EITB positivity among each lesion subtype and its association with perilesional edema were determined. There were significantly higher positive EITB results in patients with GMNCC (90/111, 81.1%) compared with other lesion types. Perilesional edema was associated with positive EITB in patients with CMNCC. On univariate analysis, perilesional edema and GMNCC were associated with EITB positivity. On multivariate analysis, only GMNCC (OR 7.5; 95% CI 3.5 to 16.2) was significantly associated with EITB positivity. In patients with pNCC, the presence of perilesional edema is associated with a higher probability of a positive EITB result in patients with CMNCC, suggesting a synchronicity in the mechanisms associated with formation of perilesional edema and the antibody response in this subtype. In patients with enhancing granulomas, edema is not an independent predictor of a positive EITB, suggesting that the enhancement itself is associated with a strong antibody response.

A Large Case Series of Neurocysticercosis in Kuwait, a Nonendemic Arabian Gulf Country in the Middle East Region.

Neurocysticercosis (NCC), a leading global cause of severe progressive headache and epilepsy, in developed or affluent countries is mostly diagnosed among immigrants from poor or developing Taenia solium taeniasis-endemic countries. Taeniasis carriers in Kuwait are routinely screened by insensitive stool microscopy. In this study, enzyme-linked immunoelectrotransfer blot (EITB) was used as a confirmatory test for NCC. Screening was performed on 970 patients referred for suspected NCC on the basis of relevant history and/or ring-enhancing lesions on computed tomography and/or magnetic resonance imaging during a 14-year period in Kuwait. Demographic data and clinical details were retrieved from laboratory or hospital records. EITB was positive in 150 subjects (15.5%), including 98 expatriates mostly originating from taeniasis-endemic countries and, surprisingly, 52 Kuwaiti nationals. The clinical details of 48 of 50 NCC cases diagnosed during 2014–2019 were available. Most common symptoms included seizures, persistent headache with/without fever, and fits or loss of consciousness. Cysticercal lesions were located at various brain regions in 39 of 48 patients. Multiple members of 3 families with NCC were identified; infection was linked to domestic workers from taeniasis-endemic countries and confirmed in at least 1 family. Our data show that NCC is predominantly imported in Kuwait by expatriates originating from taeniasis-endemic countries who transmit the infection to Kuwaiti citizens.

Evidence for Transmission of Taenia solium Taeniasis/Cysticercosis in a Rural Area of Northern Rwanda.

Cysticercosis is a parasitic infection caused by the metacestode larval stage (cysticercus) of Taenia solium. In humans, cysticercosis may infect the central nervous system and cause neurocysticercosis, which is responsible for over 50,000 deaths per year worldwide and is the major cause of preventable epilepsy cases, especially in low-income countries. Cysticercosis infection is endemic in many less developed countries where poor hygiene conditions and free-range pig management favor their transmission. A cross-sectional study was conducted in 680 children from a rural primary school in Gakenke district (Northern province of Rwanda). Stool samples were collected from participants and analyzed using the Kato-Katz method (KK), formol-ether concentration (FEC), and/or copro-antigen enzyme-linked immunosorbent assay (CoAg-ELISA) to detect taeniasis. Blood samples were collected and analyzed using enzyme-linked immunoelectrotransfer blot (EITB) and antigen enzyme-linked immunosorbent assay (Ag-ELISA) to detect human cysticercosis. The overall proportion of taeniasis positivity was 0.3% (2/680), and both cases were also confirmed by CoAg-ELISA. A total of 13.3% (76/572) of the children studied were positive to cysticercosis (T. solium-specific serum antibodies detected by EITB), of whom 38.0% (27/71) had viable cysticercus (T. solium antigens by Ag-ELISA). This study provides evidence of the highest cysticercosis prevalence reported in Rwanda in children to date. Systematic investigations into porcine and human cysticercosis as well as health education and hygiene measures for T. solium control are needed in Gakenke district.

Animal hydatid cyst genotypes as a potential substitute for human hydatid cyst as a source of antigen for diagnosis of zoonotichydatidosis.

The Diagnosis of hydatidosis is still an unsolved issue due to difficulties in obtaining of patient's hydatid cyst appropriate for antigen extraction. This study evaluated the suitability of HC protoscolices somatic antigens (HCPsS-Ag) fractions from animal origin to substitute that extracted from HC of patients in diagnosis of hydatidosis using enzyme-linked immunoelectro-transfer blot and Enzyme-linked immunosorbent assay (ELISA). Eight fractions in HC-G6 from patients react specifically versus HC-G6 infected patient's sera. Five of them (28, 32, 38, 59 and 89 Kilo Dalton (KDa) and two of them (28KDa and 45KDa) reacted versus HC-G1 and HC-G4 infected sheep and equine sera, respectively. Six fractions in HCPsS-Ag-G1 of sheep react versus HC-G1 sheep infected sera, four (28, 32, 52 and 58 KDa) and two of them reacted versus HC-G6 and HC-G4 infected patient and equine sera, respectively. Two fractions only in HCPsS-Ag-G4 of equine reacted versus infected human and sheep sera. This fraction displayed the same degree of ELISA value versus different infected sera with a significantly perfect classification for kappa agreement and non-statistically significant difference (p ≥ 0.05) for ELISA Optical density values of the positive samples without cross-reaction versus other parasites antibodies in sera. HCPsS-Ag from HC genotypes that developed in humans and animals as HC-G6 and HC-G1 can substitute each other for diagnosis of infection than antigens extracted from non-zoonotic HC-G4. The fraction at 28KDa is the only fraction that can be extracted from any animals HC and used in diagnosis of zoonotic hydatidosis.

Cysticercosis in Madagascar.

Cysticercosis (CC) is the most common parasitic disease of the central nervous system. It is endemic in most developing countries where pigs are raised and consumed. An overview of all available data of this parasite in Madagascar is lacking. We conducted a literature review, collecting information on published and available literature about cysticercosis in Madagascar between January 1st, 1990 and June 30th, 2020. Out of 858 publications; 61 were included, issued from peer-review indexed journals, non-indexed journals, books, Ministry reports and press releases. In Madagascar, porcine cysticercosis has been reported since 1901; human cysticercosis is highly prevalent with an overall estimated seroprevalence between 7 and 21%. Serological analysis is based on Enzyme-Linked Immunosorbent Assay (ELISA) and Enzyme-linked immunoelectrotransfer blot techniques (EITB) for confirmative testing. Neurocysticercosis (NCC) is the most common pattern of cysticercosis in Madagascar and it is reponsible for pediatric morbidity causing more than 50% of epilepsy cases. Though CT-Scan is now available and tends to be considered the gold standard for NCC diagnosis, it remains unaffordable for most Malagasy patients and implies the proposal of a diagnostic algorithm for physicians. Our review has revealed that human taeniasis and bovine cysticercosis is a considerable burden in Madagascar. A national control program has been developed aiming to decrease the seroprevalence rate from 16 to 10% in 2015. The aim of the country is now to implement a CC control and elimination program. Meanwhile, some massive cysticercosis screenings have been conducted in the capital Antananarivo to drive people's attention on this widespread infection.

Evaluation of Taenia solium cyst fluid-based enzyme linked immunoelectro transfer blot for Neurocysticercosis diagnosis in urban and highly endemic rural population of North India

BackgroundNeurocysticercosis (NCC) is infection by cestode Taenia solium/pork tapeworm. Sero-diagnosis of NCC is still a challenge. Radiological imaging (CT/MRI) are cost intensive, requires technical expertise and resource intensive. Hence, its availability is restricted in endemic zone. Existing Enzyme electro immune transfer blot (EITB) antigens are difficult to make and is not standardized for endemic population. Therefore, there is a definite need for easy and reliable EITB tool. MethodsT. solium metacestode were harvested from naturally infected swine post slaughter. The cyst fluid/vesicular fluid was aspirated and processed with ultracentrifugation and immune blot was performed with this antigen. ResultsA total of 406 cases [rural 256 (NCC 78, seizures other than NCC 108 and healthy controls 70); urban 150 (NCC 41, seizures other than NCC 59 and healthy controls 50)] were enrolled. Positive EITB (detection of band <50 kDa) was significantly associated with NCC patients of urban population only (p < 0.001) but not in rural populations (p = 0.292). However, identification of 15 kDa band had significant association with NCC both in urban and rural populations with overall sensitivity of 91.5% and specificity of 91.6%. Presence of 35 kDa band was associated with multiple NCC (p < 0.001). The study shows that 15 kDa reactive band on EITB is highly sensitive and specific for diagnosis of NCC in endemic population. ConclusionsPresence of 35 kDa band on EITB was associated with infection by multiple cysticerci. The observations demand purification of cyst fluid antigens to develop simple and easy to execute test in field studies.

Occurrence of Taenia species in pigs in slaughterhouses in Phu Tho province, northern Vietnam.

Pigs act as the intermediate hosts of the zoonotic tapeworms Taenia solium and Taenia asiatica, as well as of the non-zoonotic Taenia hydatigena. In Vietnam, human taeniasis and cysticercosis have been reported throughout the country; however, data on porcine cysticercosis are scarce. Our study aimed to estimate the prevalence of Taenia spp. in slaughtered pigs in two districts in Phu Tho, a mountainous province in northern Vietnam from where neurocysticercosis patients commonly originate. The carcasses of 399 pigs from 51 small-scale abattoirs were checked for cysticerci, while tongue, liver, masseter muscles, diaphragm and heart were sliced and examined. Retrieved cysticerci underwent polymerase chain reaction-restriction fragment length polymorphism and sequencing for species confirmation. Blood was also collected to detect antibodies by lentil lectin-purified glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP-EITB) and recombinant T24H antigen (rT24H)-EITB and circulating antigens by B158/B60 Ag-ELISA. In two pigs, T. asiatica cysticerci were found, confirming the presence of the parasite in pigs in Vietnam at a low prevalence (0.5%; 95% exact confidence interval (CI): 0-1.19%). Cysticerci of T. solium were found in none of the pigs, although one serum sample was positive for antibodies in both LLGP-EITB and rT24H-EITB. Furthermore, a high prevalence of T. hydatigena cysticercosis was observed (18.0%; 95% Wilson score CI: 14.6-22.1%). In more than half of the T. hydatigena-positive pigs, circulating antigens were detected by Ag-ELISA, confirming that this test cannot be used to diagnose T. solium cysticercosis in this region. Finally, Spirometra erinaceieuropaei was found in one pig liver. It is the first record of this zoonotic cestode species in pigs in Vietnam. Overall, the findings confirmed the complex epidemiology of Taenia spp. in pigs in Vietnam.

Further evaluation of recombinant Tsol-p27 by enzyme-linked immunoelectrotransfer blot for the serodiagnosis of cysticercosis in pigs from Mozambique

BackgroundPorcine cysticercosis has a negative impact on human health and the meat industry, as it makes infected meat unaproprieted for consuption and it is the main etiology of epileptic seizures in developing countries. There are multiple serological assays that use crude antigens with high sensitivity and specificity for the diagnosis of both porcine and human cysticercosis. Nonetheless, antigen preparation is time-consuming, needs a well-equipped laboratory and trained personnel and places those manipulating the meat at great risk for infection. New serodiagnostic approaches to the diagnosis of porcine and human cysticercosis have been directed towards the development of recombinant deoxyribonucleic acid technology for the generation of synthetic proteins that can serve as simplified, low-cost and harmless substitutes for native antigens. The aim of the present study was to further evaluate the recombinant Tsol-p27 protein as a target molecule in immunoassays for the serodiagnosis of porcine cysticercosis. From these data, we hoped to develop recommendations regarding its use in the serodiagnosis of porcine cysticercosis.ResultsWe studied a panel of 83 naturally infected pig sera from Angónia District, Mozambique, an endemic area for porcine and human cysticercosis. These sera were previously tested by antigen enzyme-linked immunosorbent assay (Ag-ELISA) to detect antigens of T. solium. The serum panel was processed by enzyme-linked immunoelectrotransfer blot (EITB) assay against the recombinant Tsol-p27 protein and the Ag-ELISA assay results were used to compare and evaluate the performance of Tsol-p27 for the diagnosis of cysticercosis. Out of 83 sera, 24 (29.0%) were positive for Tsol-p27 and 59 (71%) were negative in the same assay. From the 37 sera that tested positive to Ag-ELISA, 11 (13.3%) were positive to Tsol-p27, while from 46 sera that tested negative to Ag-ELISA, 33 (39.7%) also tested negative to Tsol-p27. The sensitivity and specificity of Tsol-p27 was 29.7% and 71.7%, respectively, while the positive predictive value and negative predictive value were 45.8% and 55.9%, respectively, as calculated using Medcalc® version 15.0 software (MedCalc Software, Ostend, Belgium).ConclusionWhile Tsol-p27 recombinant protein might be suitable for testing human sera, its performance in pigs is not acceptable, so other recombinant proteins should be evaluated alone or multiplexed.

OC 8432 EVALUATION OF AN ANTIBODY-DETECTING POINT-OF-CARE TEST FOR THE DIAGNOSIS OFTAENIA SOLIUMTAENIASIS AND NEUROCYSTICERCOSIS/CYSTICERCOSIS IN AN ENDEMIC AREA

BackgroundTaenia soliumtaeniasis/(neuro)cysticercosis is a neglected parasitic zoonosis with significant economic and public health impacts. Neurocysticercosis is responsible for 30% cases of acquired epilepsy in endemic areas. Diagnosis and case management of neurocysticercosis/taeniasis in resource-limited endemic countries is challenging. Reliable, inexpensive and easy to use diagnostic tools with sufficient sensitivity and specificity are currently not available. A new point-of-care (POC) test based on recombinant rT24H and rES33 proteins developed by the Centre for Disease Control in Atlanta (US) which combines diagnosis of taeniasis and cysticercosis has been developed, however, its performance at community level is not known. The aim of this study is therefore, to evaluate the diagnostic performance of this test in a community setting.MethodsThe study site is Mtandaza community, Sinda district, Eastern Province of Zambia. The diagnostic accuracy is being evaluated for taeniasis and (neuro) cysticercosis in 1200 randomly selected participants in a community-based study. The performance characteristics (sensitivity and specificity) for neurocysticercosis will be computed by cross-tabulating of POC results with those of the ‘neurocysticercosis diagnosis’ while a Bayesian approach will be used for cysticercosis and taeniasis to compare the performance of the index test with reference tests (enzyme-linked immuno-electrotransfer blot (EITB), B158/B60 Ag-ELISA, Ab-ELISA, Copro-Ag ELISA, PCR).ResultsPreliminary results of 505 POC tests so far conducted show that 0.8% were positive for taeniasis, 9.1% for cysticercosis and, 4.6% were invalid or unclear. Except for Copro-Ag and B158/B60 Ag-ELISA for taeniasis and cysticercosis respectively, reference tests are yet to be conducted.ConclusionResults will show the diagnostic value of the POC test and its applicability for use at community level in endemic areas. If successful, implementation of the tool will enable early detection of taeniasis and suspected neurocysticercosis cases and lead to improved patient management and treatment outcomes.

Evaluation of cross-reactivity to Taenia hydatigena and Echinococcus granulosus in the enzyme-linked immunoelectrotransfer blot assay for the diagnosis of porcine cysticercosis

BackgroundTaenia solium is an important zoonotic parasite that infects humans as definitive host (taeniasis) and pigs as intermediate host (cysticercosis). Serological diagnosis of porcine cysticercosis is limited to antigen detection using ELISA, which is known to cross-react with other Taenia species, and antibody detection using the lentil-lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB), which has not been adequately evaluated for cross-reactivity to other parasites. Field studies suggest that the GP50 diagnostic band of the LLGP EITB may cross-react to Taenia hydatigena, a common non-zoonotic parasitic infection of pigs. The objective of this study was to evaluate the specificity of the LLGP EITB assay in pigs infected experimentally with T. hydatigena and Echinococcus granulosus.ResultsTwelve three-month-old seronegative were divided into two groups; six were each given an oral challenge with a single gravid proglottid of T. hydatigena and the other six were each given an oral challenge with 50 gravid proglottids of E. granulosus. Serum samples were collected biweekly until 14 weeks when all pigs underwent a detailed necropsy. Taenia hydatigena cysticerci were found in two of six pigs from the first group. Four T. hydatigena-exposed pigs were seropositive at the GP50-band only on EITB LLGP; two of these had cysts at necropsy while no seronegative pigs had cysts. One E. granulosus-exposed pig was positive to EITB LLGP, again with reactivity only to GP50; all six pigs had hepatic echinococcosis on necropsy.ConclusionThese results provide definitive evidence that the GP50 diagnostic band in pigs cross-reacts with T. hydatigena. Evidence of cross-reaction with E. granulosus was not conclusive.