Contribution of IgG avidity and PCR for the early diagnosis of toxoplasmosis in pregnant women from the North-Eastern region of Algeria

Background: Acute toxoplasmosis in pregnant women presents a high risk of Toxoplasma transmission to the fetus. Early diagnosis is difficult, especially when serological testing for IgG/IgM antibodies fail to differentiate between a recent and a past infection. In this case, we rely on IgG avidity or PCR assays.Objectives: The aim of this study was to compare conventional ELISA and IgG avidity, with PCR using B1 and P30 primers for the early diagnosis of toxoplasmosis in pregnant women.Methods: Sera were collected from 143 pregnant women and measured by ELISA for anti-Toxoplasma IgG, IgM, IgA and IgG avidity. DNA was extracted from 57 peripheral blood and 14 amniotic fluid samples for PCR amplification.Results: A total of 57 out 143 women were seropositive: 30 (52.6%) were IgG+/IgM- and 27 (43.8%) were IgG+/IgM+; IgA antibodies were positive in 7 (12.2%) cases. IgG avidity was low in 9 women suggesting an acute infection; 3 women presented an intermediate avidity. PCR detected Toxoplasma DNA in 9 women presenting low avidity and was negative for the intermediate avidity cases.Conclusion: PCR combined to avidity IgG performed better than ELISA IgG, IgM and/or IgA assays alone. PCR was useful in the case of intermediate avidity.Keywords: Toxoplasmosis, pregnant women, serology, IgG avidity, PC

Anti-asialoglycoprotein receptor autoantibodies, detected by a capture-immunoassay, are associated with autoimmune liver diseases.

In autoimmune chronic active hepatitis (AIH) and primary biliary cirrhosis (PBC), various autoantibodies including anti-asialoglycoprotein receptor (ASGPR) antibodies have been found in patients' sera. We have previously developed a mouse monoclonal antibody against rat and human ASGPR. In this study, we developed a capture enzyme-linked immunosorbent assay (ELISA) for detection of anti-ASGPR antibodies using this monoclonal antibody and investigated the occurrence of anti-ASGPR antibodies in the sera of patients with various liver diseases. Serum samples were obtained from 123 patients with various liver diseases, including 21 patients with AIH and 40 patients with PBC. In this capture ELISA, the target antigen in the crude rat liver membrane extracts was captured on the ELISA wells by the ASGPR-specific mouse monoclonal antibody. Thus, the cumbersome process of antigen purification was rendered unnecessary. Using this capture ELISA, we detected the anti-ASGPR antibody in 67% of the patients with AIH, in 100% of the patients with PBC, and in 57% of the patients with acute hepatitis type A. However, the anti-ASGPR antibody was rarely detected in patients with other liver diseases such as primary sclerosing cholangitis and obstructive jaundice. Our findings suggest that this capture ELISA would be useful for the detection of anti-ASGPR antibodies in autoimmune liver diseases.</p

Determination of carbendazim residues in Moroccan tomato samples using local enzyme-linked immunosorbent assay and comparison with liquid chromatography

The fungicide carbendazim (CBZ) is not approved for agricultural uses in some countries but is still used by many farmers due to its effectiveness. For this reason, in previous work of the same authors, they developed a competitive enzyme immunoassay (ELISA) using rabbit polyclonal antibodies to detect CBZ. This study aimed to validate this in-house ELISA after extraction with methanol for CBZ analysis in tomato samples, and the results were compared with the conventional high-performance liquid chromatography (HPLC) method after QuEChERS extraction. The results showed that both ELISA and HPLC methods have good repeatability, reproducibility and high precision with a good variation verified by principal components analysis (PCA). ANOVA tested the detection limit (LOD), and quantification limit (LOQ), and the values for ELISA (LOD = 0.026± 0.001 µg/L and LOQ = 0.083 ± 0.003 µg/L) were significantly lower than those obtained by HPLC (LOD = 0.61 ± 0.02 µg/L and LOQ = 1.85 ± 0.07 µg/L). ELISA and HPLC were used for analyzing CBZ in 100 Moroccan tomato samples. These two methods detected the presence of CBZ above the Maximum Residue Limit (MRL) level in 9 samples. However, the presence of the  CBZ was detected in the 79 samples by ELISA and quantified in 66 samples. In contrast, the presence of CBZ was detected in 57 and quantified in 35 samples by HPLC. These results showed that the ELISA system coupled with a simple methanol extraction is much more sensitive than HPLC after QuEChERS extraction

Influence of infection on malaria-specific antibody dynamics in a cohort exposed to intense malaria transmission in northern Uganda.

The role of submicroscopic infections in modulating malaria antibody responses is poorly understood and requires longitudinal studies. A cohort of 249 children ≤5 years of age, 126 children between 6 and 10 years and 134 adults ≥20 years was recruited in an area of intense malaria transmission in Apac, Uganda and treated with artemether/lumefantrine at enrolment. Parasite carriage was determined at enrolment and after 6 and 16 weeks using microscopy and PCR. Antibody prevalence and titres to circumsporozoite protein, apical membrane antigen-1 (AMA-1), merozoite surface protein-1 (MSP-119 ), merozoite surface protein-2 (MSP-2) and Anopheles gambiae salivary gland protein 6 (gSG6) were determined by ELISA. Plasmodium falciparum infections were detected in 38·1% (194/509) of the individuals by microscopy and in 57·1% (284/493) of the individuals by PCR at enrolment. Antibody prevalence and titre against AMA-1, MSP-119 , MSP-2 and gSG6 were related to concurrent (sub-)microscopic parasitaemia. Responses were stable in children who were continuously infected with malaria parasites but declined in children who were never parasitaemic during the study or were not re-infected after treatment. These findings indicate that continued malaria infections are required to maintain antibody titres in an area of intense malaria transmission

Specific immunoassays confirm association of <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> with type-1 but not type-2 diabetes mellitus

Background Mycobacterium avium subspecies paratuberculosis (MAP) is a versatile pathogen with a broad host range. Its association with type-1 diabetes mellitus (T1DM) has been recently proposed. Rapid identification of infectious agents such as MAP in diabetic patients at the level of clinics might be helpful in deciphering the role of chronic bacterial infection in the development of autoimmune diseases such as T1DM. Methodology/Principal Findings We describe use of an ELISA method to identify live circulating MAP through the detection of a cell envelope protein, MptD by a specific M13 phage – fMptD. We also used another ELISA format to detect immune response to MptD peptide. Both the methods were tested with blood plasma obtained from T1DM, type-2 diabetes (T2DM) patients and non-diabetic controls. Our results demonstrate MptD and fMptD ELISA assays to be accurate and sensitive to detect MAP bacilli in a large fraction (47.3%) of T1DM patients as compared to non-diabetic controls (12.6%) and those with confirmed T2DM (7.7%). Comparative analysis of ELISA assays performed here with 3 other MAP antigen preparations, namely HbHA, Gsd and whole cell MAP lysates confirmed comparable sensitivity of the MptD peptide and the fMptD based ELISA assays. Moreover, we were successful in demonstrating positive bacterial culture in two of the clinical specimen derived from T1DM patients. Conclusions and Significance The MptD peptide/fMptD based ELISA or similar tests could be suggested as rapid and specific field level diagnostic tests for the identification of MAP in diabetic patients and for finding the explanations towards the occurrence of type-1 or type-2 diabetes in the light of an active infectious trigger

Novel screening methods for the detection of Yersinia enterocolitica in infected blood used for transfusion : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

Between 1991-1996, 8 patients experienced rare life-threatening reactions that followed the transfusion of blood infected with Yersinia enterocolitica. The first reported case occurred in 1991 and was followed by seven others that directly caused or contributed to the death of 5 of 8 patients. Y. enterocolitica is a food and water borne infection of the gastrointestinal tract which in adults is often asymptomatic. An unknown number of those infected experience a period of self-limiting bacteraemia. The large volume of blood collected during donation phlebotomy may contain small numbers of bacteria that can increase in number during blood bank storage, producing potentially lethal levels of bacteria and toxin. Currently there are no reliable methods available to distinguish blood donations that present the greatest risk from those that present little risk. This thesis, reports on the evaluation of two techniques to prevent the transfusion of blood infected with Y. enterocolitica. The first, a molecular method, was used to amplify bacterial DNA in blood by Polymerase Chain Reaction (PCR). A 425 bp product was amplified from DNA extractions of infected blood. Results showed that the technical complexities of the methodology, together with poor sensitivity and the need for large-scale donation sampling make PCR as applied for this purpose unattractive. An Enzyme Linked Immunosorbent Assay was developed to detect current/recent infection with Y. enterocolitica in healthy blood donors. Polystyrene beads were coated with bacterial proteins to detect IgA antibody to Y. enterocolitica in human serum. The sera from donors of confirmed unit infections, paired sera from culture-proven Y. enterocolitica gastrointestinal tract infection and sera from volunteer blood donors were tested. Results showed that the sera of six bacteraemic blood donors tested contained elevated levels of IgA antibody. High rates of positivity (26/27), were detected in sera from culture-confirmed GIT infection and a rate of 4.04% seropositivity was found among 495 blood donors enrolled in a clinical trial. Results showed a strong correlation between IgA seropositivity, and recipient risk associated with the transfusion of blood heavily infected with Y. enterocolitica. The work demonstrated how the use of a simple screening test for recent infection, could be used to exclude high risk donations and improve the safety of blood transfusion in New Zealand

Alemtuzumab preconditioning with tacrolimus monotherapy - The impact of serial monitoring for donor-specific antibody

BACKGROUND. Antibody preconditioning with tacrolimus monotherapy has allowed many renal allograft recipients to be maintained on spaced weaning. METHODS. Of 279 renal allograft recipients transplanted between March 2003 and December 2004, 222 (80%) had spaced weaning (i.e., reduction of tacrolimus monotherapy dosing to every other day, three times a week, twice a week, or once a week) attempted. Routine monitoring for donor-specific antibody (DSA) was begun in September 2004. Mean follow-up is 34±6.5 months after transplantation and 26±8.1 months after the initiation of spaced weaning. RESULTS. One hundred and twenty-two (44%) patients remained on spaced weaning. One- and 2-year actual patient/graft survival was 99%/99%, and 97%/96%. Fifty-six (20%) patients experienced acute rejection after initiation of spaced weaning. One- and 2-year actual patient/graft survival was 100%/98%, and 94%/78%. Forty-two (15%) patients with stable renal function had spaced weaning stopped because of the development of DSA, which disappeared in 17 (40%). One- and 2-year actual patient and graft survival was 100% and 100%. CONCLUSION. Adult renal transplant recipients who are able to be maintained on spaced weaning have excellent outcomes. Patients with stable renal function who have reversal of weaning because of the development of DSA also have excellent outcomes. Routine monitoring for DSA may allow patients to avoid late rejection after spaced weaning. © 2008 Lippincott Williams & Wilkins, Inc

Rapid Detection of Leishmania infantum Infection in Dogs: Comparative Study Using an Immunochromatographic Dipstick Test, Enzyme-Linked Immunosorbent Assay, and PCR

Current zoonotic visceral leishmaniasis (ZVL) control programs in Brazil include the culling of Leishmania infantum-infected reservoir dogs, a strategy that has failed to prevent a rise of canine and human ZVL cases over the past decade. One of the main reasons this strategy has failed is because of a long delay between sample collection, sample analysis, and control implementation. A rapid, sensitive, and specific diagnostic tool would be highly desirable, because it would allow control interventions to be implemented in situ. We compared an immunochromatographic dipstick test to enzyme-linked immunosorbent assay (ELISA) and PCR for detecting L. infantum infections in dogs from an area of ZVL endemicity in Brazil. The dipstick test was shown to have 61 to 75% specificity and 72 to 77% sensitivity, compared to 100% specificity for both ELISA and PCR and 71 to 88% and 51 to 64% sensitivity for ELISA and PCR, respectively. Of the field samples tested, 92 of 175 (53%), 65 of 175 (37%), and 47 of 175 (27%) were positive by dipstick, ELISA, and PCR, respectively. The positive and negative predictive values for the tested dipstick were 58 to 77% and 75%, respectively. Efforts should be made to develop a more specific dipstick test for diagnosis of leishmaniasis, because they may ultimately prove more cost-effective than currently used diagnostic tests when used in mass-screening surveys

Electrochemical detection of Toxocara canis excretory-secretory antigens in children from rural communities in Esmeraldas Province, Ecuador: association between active infection and high eosinophilia.

BACKGROUND: The diagnosis of active Toxocara canis infections in humans is challenging. Larval stages of T. canis do not replicate in human tissues and disease may result from infection with a single T. canis larva. Recently, we developed a nanobody-based electrochemical magnetosensor assay with superior sensitivity to detect T. canis excretory-secretory (TES) antigens. Here, we evaluate the performance of the assay in children from an Ecuadorian birth cohort that followed children to five years of age. METHODS: Samples were selected based on the presence of peripheral blood eosinophilia and relative eosinophil counts. The samples were analyzed by the nanobody-based electrochemical magnetosensor assay, which utilizes a bivalent biotinylated nanobody as capturing agent on the surface of streptavidin pre-coated paramagnetic beads. Detection was performed by a different nanobody chemically labelled with horseradish peroxidase. RESULTS: Of 87 samples tested, 33 (38%) scored positive for TES antigen recognition by the electrochemical magnetosensor assay. The average concentration of TES antigen in serum was 2.1 ng/ml (SD = 1.1). The positive result in the electrochemical assay was associated with eosinophilia > 19% (P = 0.001). Parasitological data were available for 57 samples. There was no significant association between positivity by the electrochemical assay and the presence of other soil-transmitted helminth infections. CONCLUSIONS: Our nanobody-based electrochemical assay provides highly sensitive quantification of TES antigens in serum and has potential as a valuable tool for the diagnosis of active human toxocariasis