Dataset Information

A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.

ABSTRACT: Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, we have developed a new copro-ELISA test after extensive screening of a large panel of monoclonal antibodies (MAbs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. The key component of the test, MAbEg9 has a convenient IgG isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. Time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. The test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. This characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in Peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion.

SUBMITTER: Morel N

PROVIDER: S-EPMC3542170 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.

Morel Noelia N Lassabe Gabriel G Elola Susana S Bondad Mauricio M Herrera Silvia S Marí Carlos C Last Jerold A JA Jensen Oscar O Gonzalez-Sapienza Gualberto G

PLoS neglected tropical diseases 20130110 1

Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, w ...[more]

PMID: 23326610

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Project description:ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1-10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis.

Project description:Intestinal capillariasis is a parasitic zoonosis caused by the tiny nematode parasite Capillaria philippinensis. It is a major health problem that may lead to death if not diagnosed and treated appropriately. The difficulties in the diagnosis of C. philippinensis highlight the importance of developing accurate, sensitive, and specific methods for early diagnosis. This study aimed to detect the presence of C. philippinensis infection among 42 clinically suspected patients with certain criteria that are highly suggestive of capillariasis and to compare the diagnostic yield of microscopy, copro-ELISA, and PCR for the detection of copro-DNA. Sociodemographic characteristics and clinical data were also described for the infected group. Out of 42 patients, 10 were microscopically positive, 40 samples were positive by copro-ELISA, nested PCR detected 35 positive cases, with total detection rates of 23.8%, 95.2%, and 83.3% using direct microscopic examination, copro-ELISA, and PCR, respectively. The majority of positive cases were females, middle-aged people, and people from rural areas. The real number of cases infected with C. philippinensis may far exceed those estimated using microscopy. The diagnosis by copro-ELISA for the detection of C. philippinensis coproantigen and by nested PCR to identify parasite DNA revealed a higher number of positive cases. Using ELISA for the detection of coproantigen is a sensitive test that identifies the infection, yet it is not specific. Copro-DNA offers a satisfactory sensitive and specific method for the detection of infection in clinically suspected patients. The most susceptible individuals to C. philippinensis infection are females, middle-aged people, and people of low social standards. Intestinal capillariasis needs to be considered in patients who present with symptoms of chronic diarrhea and hypoalbuminemia because if these cases are left undiagnosed and untreated, they may suffer from lethal complications.

Project description:Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed.We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages.We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis.Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.

Dataset Information

A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.

Publications

A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.

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