Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena.

BACKGROUND: Antigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA. METHODS: We report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland-Altman (BA) analysis and Lin's concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA. RESULTS: The TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin's CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin's CCC: 0.24) but increased on days 28 and 90 PI (Lins' CCC: 0.88 and 0.98, P < 0.001). CONCLUSIONS/SIGNIFICANCE: The TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.

Consistent Measurement of Parasite-Specific Antigen Levels in Sera of Patients with Neurocysticercosis Using Two Different Monoclonal Antibody (mAb)-Based Enzyme-Linked Immunosorbent Assays.

Monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) is a complementary diagnosis technique for neurocysticercosis (NCC), which detects circulating parasite antigen (Ag) indicative of viable infection and Ag levels that correlate well with the parasite burden. In this study, we compared the performance of two Ag-ELISA techniques for the detection of NCC. We assessed the agreement between our in-house TsW8/TsW5 Ag-ELISA and the widely used B158/B60 Ag-ELISA for measuring T. solium antigen levels in the sera from 113 patients with calcified, parenchymal, and subarachnoid NCC. Concordance was demonstrated evaluating the limits of agreement (LoAs) stratified by the type of NCC. Both ELISA's detected 47/48 (97.8%) subarachnoid NCC cases. In parenchymal and calcified NCC, the B158/B60 Ag-ELISA detected 19/24 (79.2%) and 18/41 (43.9%) cases, while the TsW8/TsW5 Ag-ELISA detected 21/24 (87.5%) and 13/41 (31.7%), respectively. Parenchymal and calcified NCC obtained a perfect agreement (100%), indicating that all sample results were within the predicted LoA, while for subarachnoid NCC, the agreement was 89.6%. The high concordance between the assays was confirmed by Lin's concordance coefficient (LCC = 0.97). Patients with viable parenchymal NCC (LCC = 0.95) obtained the highest concordance between assays, followed by subarachnoid NCC (LCC = 0.93) and calcified NCC (LCC = 0.92). The TsW8/TsW5 Ag-ELISA and B158/B60 Ag-ELISA showed high Ag measurement correlations across diverse types of NCC.

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.

Population Screening for Urine Antigens to Detect Asymptomatic Subarachnoid Neurocysticercosis: A Pilot Study in Northern Peru.

Subarachnoid neurocysticercosis (SANCC) is a severe and progressive brain infection with Taenia solium. We performed a pilot study of noninvasive screening for SANCC in two endemic villages in northern Peru using a urine antigen screen followed by brain magnetic resonance imaging for participants with elevated levels of antigen. Among the 978 participants screened, we identified eight individuals with SANCC, many of whom were asymptomatic. This represents a minimum prevalence of 0.8% of SANCC, a level higher than expected based on prior studies, and a positive predictive value of 62% for our novel urine screening test. Future studies should confirm whether early detection and management improve clinical outcomes.

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

BACKGROUND: Taenia 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. RESULTS: Twelve 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. CONCLUSION: These 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.

Performance of a Sandwich Antigen-Detection ELISA for the Diagnosis of Porcine Taenia solium Cysticercosis.

The pig is the natural intermediate host of Taenia solium, a parasite causing significant burden of disease in both humans and pigs. Porcine cysticercosis is traditionally detected via tongue palpation and slaughterhouse meat inspection, both with limited sensitivity. Serum antibody detection has a better performance; however, it does not discriminate past from present infection. Serum antigen detection can demonstrate viable infection and gives a good estimate of parasitic load. This study evaluated a sandwich antigen-detection ELISA using monoclonal antibodies (MoAbs) 158C11 and 60H8 for the diagnosis of viable cysticercosis in pigs. Serum samples were used from 35 naturally T. solium cysticerci-infected pigs, 31 cysticercosis-negative pigs, and 22 pigs with Taenia hydatigena infection (to assess cross-reactions). Positive cysticercosis samples were subcategorized at necropsy according to parasitic burden as mild (1-10 viable cysts, n = 10), moderate (11-100 cysts, n = 5), or severe infection (more than 100 cysts, n = 20). This Ag-ELISA showed a sensitivity of 82.9% and a specificity of 96.8% when not considering cross-reactions with T. hydatigena. Hundred percentage of severely infected, 80% of moderately infected, and 50% of mildly T. solium-infected pigs tested positive. Twenty of 22 pigs with only T. hydatigena infections were positive, with 13 reaching saturating levels in the ELISA. The Ag-ELISA revealed the presence of live cysts and is, thus, a fairly reliable test to monitor experimental infection, response to treatment, and follow-up in animal models of cysticercosis. It should, however, be carefully interpreted when used in regions where T. hydatigena is endemic in pigs.

Epidemiology of Taenia saginata taeniosis/cysticercosis: a systematic review of the distribution in the Americas.

BACKGROUND: The distribution of Taenia saginata in the Americas is unclear. Establishing the distribution, economic burden, and potentials for control of bovine cysticercosis is increasingly important due to the growing demand for beef. This paper aims to take the first step and reviews the recent distribution of T. saginata taeniosis and bovine cysticercosis on a national level within the Americas. METHODS: We undertook a systematic review of published and grey literature for information on the occurrence, prevalence, and geographical distribution of bovine cysticercosis and human taeniosis in the 54 countries and territories of the Americas between January 1st, 1990 and December 31st, 2017. Data on bovine cysticercosis from OIE reports from 1994 to 2005 were also included. RESULTS: We identified 66 papers from the Americas with data on the occurrence of taeniosis or bovine cysticercosis and an additional 19 OIE country reports on bovine cysticercosis. Taeniosis was reported from 13 countries, with nine of these countries reporting specifically T. saginata taeniosis, and four countries reporting non-species specific taeniosis. The reported prevalence of taeniosis ranged between 0.04-8.8%. Bovine cysticercosis was reported from 19 countries, nine identified through the literature search, and an additional 10 identified through the OIE country reports for notifiable diseases. The reported prevalence of bovine cysticercosis ranged between 0.1-19%. Disease occurrence was restricted to 21 countries within the Americas, the majority from the mainland, with the only island nations reporting either bovine cysticercosis or taeniosis being Cuba, Haiti, and the US Virgin Islands. CONCLUSIONS: Taenia saginata is widely distributed across 21 of the 54 countries in the Americas, but insufficient epidemiological data are available to estimate the subnational spatial distribution, prevalence, incidence and intensity of infections. This needs to be addressed through active surveillance and disease detection programmes. Such programmes would improve the data quantity and quality, and may enable estimation of the economic burden due to bovine cysticercosis in the region in turn determining the requirement for and cost-effectiveness of control measures.

Spontaneously Arrested Transmission of Cysticercosis in a Highly Endemic Village with a Very Low Migration Rate.

Taenia solium cysticercosis is difficult to eliminate without interventions or societal development. Atahualpa is a rural Ecuadorian village with documented low migration rate, where domestic pig raising is common and human cysticercosis is endemic. To assess neurocysticercosis (NCC) prevalence, 1,273 villagers aged ≥ 20 years underwent neuroimaging studies, which showed calcified lesions in 121 (9.5%) individuals, but no active disease. Likewise, positive reactions, apparently nonspecific, were found in only 3/200 subjects by the use of a monoclonal antibody-based enzyme-linked immunosorbent assay to detect T. solium antigens in urine. Only 2/418 pigs reacted to three antibody bands on serum western blot and none to more than three bands. This is the first time that spontaneously arrested T. solium transmission is documented in a known endemic village. Understanding why active transmission stopped could provide insights on potential targets for control interventions. Atahualpa could provide an optimal scenario for longitudinal studies on the consequences of calcified NCC.

Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena-infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

Distribution and Potential Indicators of Hospitalized Cases of Neurocysticercosis and Epilepsy in Ecuador from 1996 to 2008.

BACKGROUND: Epilepsy is one of the most common signs of Neurocysticercosis (NCC). In this study, spatial and temporal variations in the incidence of hospitalized cases (IHC) of epilepsy and NCC in Ecuadorian municipalities were analyzed. Additionally, potential socio-economic and landscape indicators were evaluated in order to understand in part the macro-epidemiology of the Taenia solium taeniasis/cysticercosis complex. METHODOLOGY: Data on the number of hospitalized epilepsy and NCC cases by municipality of residence were obtained from morbidity-hospital systems in Ecuador. SatScan software was used to determine whether variations in the IHC of epilepsy and NCC in space and time. In addition, several socio-economic and landscape variables at municipality level were used to study factors intervening in the macro-epidemiology of these diseases. Negative Binomial regression models through stepwise selection and Bayesian Model Averaging (BMA) were used to explain the variations in the IHC of epilepsy and NCC. PRINCIPAL FINDINGS: Different clusters were identified through space and time. Traditional endemic zones for NCC and epilepsy, recognized in other studies were confirmed in our study. However, for both disorders more recent clusters were identified. Among municipalities, an increasing tendency for IHC of epilepsy, and a decreasing tendency for the IHC of NCC were observed over time. In contrast, within municipalities a positive linear relationship between both disorders was found. An increase in the implementation of systems for eliminating excrements would help to reduce the IHC of epilepsy by 1.00% (IC95%; 0.2%-1.8%) and by 5.12% (IC95%; 3.63%-6.59%) for the IHC of NCC. The presence of pig production was related to IHC of NCC. CONCLUSION/SIGNIFICANCE: Both disorders were related to the lack of an efficient system for eliminating excrements. Given the appearance of recent epilepsy clusters, these locations should be studied in depth to discriminate epilepsies due to NCC from epilepsies due to other causes. Field studies are needed to evaluate the true prevalence of cysticercosis in humans and pigs in different zones of the country in order to better implement and manage prevention and/or control campaigns.

Incidence of human Taenia solium larval Infections in an Ecuadorian endemic area: implications for disease burden assessment and control.

BACKGROUND: Human cysticercosis is a zoonotic disease causing severe health disorders and even death. While prevalence data become available worldwide, incidence rate and cumulative incidence figures are lacking, which limits the understanding of the Taenia solium epidemiology. METHODOLOGY/PRINCIPAL FINDINGS: A seroepidemiological cohort study was conducted in a south-Ecuadorian community to estimate the incidence rate of infection with and the incidence rate of exposure to T. solium based on antigen and antibody detections, respectively. The incidence rate of infection was 333.6 per 100,000 person-years (95% CI: [8.4-1,858] per 100,000 person-years) contrasting with a higher incidence rate of exposure 13,370 per 100,000 person-years (95% CI: [8,730-19,591] per 100,000 person-years). The proportion of infected individuals remained low and stable during the whole study year while more than 25% of the population showed at least one antibody seroconversion/seroreversion during the same time period. CONCLUSIONS/SIGNIFICANCE: Understanding the transmission of T. solium is essential to develop ad hoc cost-effective prevention and control programs. The estimates generated here may now be incorporated in epidemiological models to simulate the temporal transmission of the parasite and the effects of control interventions on its life cycle. These estimates are also of high importance to assess the disease burden since incidence data are needed to make regional and global projections of morbidity and mortality related to cysticercosis.

Immunological and molecular diagnosis of cysticercosis.

Cysticercosis, the infection with the larval stage of Taenia solium, is a cause of neurological symptoms including seizures, affecting the quality of life of patients and their families. Diagnosis focuses on brain imaging and serological tests are mostly used as confirmatory tools. Most cases, however, occur in poor endemic areas, where both kinds of diagnostic tools are poorly available. Development of point of care diagnostic tests is one of the most important priorities for cysticercosis researches today. The ideal point of care test would require detection of viable cysticercosis and hopefully identify cases with severe or progressive forms of neurocysticercosis, leading to referral of the patient for specialized medical attention. This manuscript describes the evolution of the serological diagnosis of cysticercosis over time, and the characteristics of the most common currently available tools, their advantages and disadvantages, and their potential use in future diagnostic tests.

Infection with versus exposure to Taenia solium: what do serological test results tell us?

Taenia solium cysticercosis is an endemic zoonosis in many developing countries. Serological tests are the most appropriate diagnostic tools to understand the transmission dynamics of the parasite, but the performances of these methods in such a setting are not known. A south Ecuadorian human population living in an endemic area was tested using three common serological tests. Because none of them is a gold standard, a Bayesian Latent Class analysis was used to estimate the test characteristics. Two definitions of a case were considered to differentiate between prevalence of current infection and prior exposure to the parasite. Differences between the performances of the same test in function of the definition of a case were observed. This study shows that test results and prior information should be interpreted carefully in a Bayesian analysis framework, particularly when the latter is based on clinical studies.

Detection of Taenia solium antigens and anti-T. solium antibodies in paired serum and cerebrospinal fluid samples from patients with intraparenchymal or extraparenchymal neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is a frequent cause of epilepsy worldwide. Compared with the more common parenchymal brain cysts, extraparenchymal infections are difficult to manage and have a poor prognosis. Serological assays are used to detect circulating Taenia solium antigens or anti-T. solium antibodies in serum or cerebrospinal fluid (CSF) samples. There are no guidelines on whether to use serum or CSF specimens for a particular assay. METHODS: We obtained paired serum and CSF samples from 91 patients with NCC (48 had intraparenchymal NCC, and 43 had extraparenchymal NCC) for detection of antibodies, using an enzyme-linked immunotransfer blot (EITB) assay, and antigens, using a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA). RESULTS: For the intraparenchymal NCC group, the EITB assay yielded more true-positive results for serum samples, and the ELISA yielded slightly more true-positive results for CSF samples than for serum samples, but none of these differences were statistically significant. Most patients with calcified NCC were antibody positive but antigen negative. For extraparenchymal disease, all samples were antibody positive, and all but 2 were antigen positive, with most samples containing high antigen levels. CONCLUSIONS: The sensitivity of antibody-detecting EITB assays is not increased through the use of CSF samples rather than serum samples. The antigen-detecting ELISA performed better for CSF samples than for serum samples, but for both specimen types it was less sensitive than the EITB assay. Active and inactive NCC are better differentiated from each other by the antigen-detecting ELISA, for both serum and CSF samples. High antigen levels suggest the presence of subarachnoid NCC.

Urine antigen detection for the diagnosis of human neurocysticercosis.

Neurocysticercosis (NCC) is a major cause of seizures and epilepsy. Diagnosis is based on brain imaging, supported by immunodiagnosis in serum or cerebrospinal fluid (CSF). Lumbar puncture is invasive and painful. Blood sampling is slightly painful and poorly accepted. Urine antigen detection has been used for other parasites and tried in NCC with suboptimal performance. We used a monoclonal antibody-based ELISA to detect Taenia solium antigens in urine from 87 Peruvian neurocysticercosis patients (viable cysts, N = 34; subarachnoid cysticercosis, N = 10; degenerating parasites, N = 7; calcified lesions, N = 36) and 32 volunteers from a non-endemic area of Peru. Overall sensitivity of urine antigen detection for viable parasites was 92%, which decreased to 62.5% in patients with a single cyst. Most patients (30/36, 83%) with only calcified cysticercosis were urine antigen negative. Antigen levels in paired serum/urine samples (evaluated in 19 patients) were strongly correlated. Non-invasive urine testing for T. solium antigens provides a useful alternative for NCC diagnosis.

Preliminary observations on Mycobacterium spp. in dairy cattle in Ecuador.

This study evaluated bovine tuberculosis in Mejia canton, a major dairy cattle production region in Ecuador. Randomly selected cattle (1,012 from 59 farms) classified according to herd size were tested by the single tuberculin test (STT). Sixty days later, positive reactors were tested again by the comparative tuberculin test (CTT). In addition, tissue samples from two STT-CTT-positive reactors detected on a farm were obtained in a local slaughterhouse and analyzed bacteriologically. A total of 4.24% of the cattle were positive in the STT and 3.85% were positive in the CTT, with the highest number (7.95%) in large herds versus 3.4% in medium herds and 0.3% in small herds. Mycobacterium bovis was isolated from mesenteric lymph nodes and lungs of one animal. A 16S ribosomal RNA-based polymerase chain reaction confirmed culture results and differentiated mycobacteria other than M. tuberculosis. This study confirms the zoonotic importance of tuberculosis in Ecuadorian dairy cattle with herd size likely to be a crucial parameter in the prevalence of the disease. The implementation of a national control program is necessary and should be based on the detection of positive cattle by STT in combination with CTT.