IgG Avidity ELISA Research Articles

Controlling Endemic Neospora caninum-Related Abortions in a Dairy Herd From Argentina.

After diagnosis of endemic abortions due to neosporosis in a commercial dairy farm, routes of Neospora caninum-transmission were evaluated in order to choose the best strategy for reducing its seroprevalence and related abortions. Fifty two dam-calf pairs were bled at parturition. Additionally, 22 female calves were also sampled at regular 3 month intervals until 18–22 months. N. caninum specific antibodies were assayed by IFAT. Serum samples were tested at a dilution 1:25 for calves before colostrum intake and heifers before mating and 1:100 for multiparous cows. Only serum samples from IFAT seropositive cattle involved in the evaluation of the routes of transmission were assessed by a commercial IgG avidity ELISA. Seropositive cows or heifers were artificially inseminated with semen from Hereford bulls. The progenies from these female animals were sent to a feed lot to produce meat. Different generalized linear models (GLM) were used to study the relationship between abortion, age category, and serostatus. Seropositive heifers were more likely to have a record of abortion (OR 2.7; 95% CI 1.6–4.7). Vertical transmission frequency was 55.5% (5 seropositive calves/9 seropositive cows). Horizontal transmission was 22.7% (5 female calves seroconverted at least one time/22 females calves sampled during 24 months) and these 5 female calves had low avidity. In heifers, both seroprevalence and abortion rates decreased from 22.1 and 8.4% of 475 in 2009 to 6.1 and 4.3% of 578 in 2015, respectively (p < 0.01). Over 5 years, N. caninum-seroprevalence and the related abortions in heifers decreased after the control strategy was assessed.

Seroprevalence of toxoplasmosis in voluntary blood donors of Puducherry and surrounding districts of Tamil Nadu.

Our objective is to study the seroprevalence of toxoplasmosis in the voluntary blood donors of Puducherry and surrounding districts of Tamil Nadu. A total of 275 healthy blood donors were screened for the presence of IgM and IgG antibodies to Toxoplasma gondii by ELISA test. Donor samples positive for IgM and/or IgG antibodies to T. gondii were subjected to IgG avidity ELISA. While, 54 out of 275 donors had IgG antibodies (19.66%), only one donor had IgM (0.36%) along with IgG. Among 54 IgG positive donors, only two had low avidity (3.7%), indicating recent exposure to the protozoa. Feasibility and cost effectiveness studies should be conducted throughout India to decide regarding screening of blood donors for toxoplasmosis.

Identification, production and assessment of two Toxoplasma gondii recombinant proteins for use in a Toxoplasma IgG avidity assay

The IgG avidity assay is an important tool in the management of suspected toxoplasmosis in pregnant women. This study aimed to produce new Toxoplasma gondii recombinant proteins and to assess their usefulness in an IgG avidity assay. Toxoplasma positive and negative serum samples were used, the former were categorized into low (LGA) and high (HGA) IgG avidity samples. Immunoblots were performed on 30 T. gondii cDNA clones to determine the reactivity and IgG avidity to the expressed proteins. Two of the clones were found to have diagnostic potential and were analyzed further; AG12b encoded T. gondii apical complex lysine methyltransferase (AKMT) protein and AG18 encoded T. gondii forkhead-associated (FHA) domain-containing protein. The His-tagged recombinant proteins, rAG12b and rAG18, were expressed and tested with LGA and HGA samples using an IgG avidity western blot and ELISA. With the IgG avidity western blot, rAG12b identified 86.4% of LGA and 90.9% of HGA samples, whereas rAG18 identified 81.8% of both LGA and HGA samples. With the IgG avidity ELISA, rAG12b identified 86.4% of both LGA and HGA samples, whereas rAG18 identified 77.3% of LGA and 86.4% of HGA serum samples. This study showed that the recombinant antigens were able to differentiate low avidity and high avidity serum samples, suggesting that they are potential candidates for use in the Toxoplasma IgG avidity assay.

APPRAISAL OF PRENATAL ANTI-TOXOPLASMA GONDII (IGG+IGM)- IHA/IGM-ELISA SCREENING IN SINGLE SAMPLES VIA IGG AVIDITY TEST.

Congenital toxoplasmosis is associated with important morbidity and mortality. Since vertical transmission of Toxoplasma gondii can occur in acute cases, antenatal screening for recent infections is vital. Accurate determination of acute toxoplasmosis requires a combination of immunoassays, usually not routinely applied for screening purposes. This study evaluated the anti-T. gondii (IgG+IgM)/IgM prenatal screening procedure by IgG avidity assay. The routine prenatal screening for (IgG+IgM) anti-T. gondii by indirect hemagglutination (IHA) in serum samples was done of 2247 pregnant women who attended two hospitals between 2011 and 2013 revealed 487 (21.7%) positive samples. Examination of IHA-positive sera by IgM and IgG/IgG-avidity concurrent ELISA tests revealed 7 positive and 3 border-line IgM-ELISA titers during the initial check-up of 10 women, who were then followed up at 3-4 week-intervals. Among these, 4 (40%) showed simultaneous high avidity IgG antibodies, indicating distant infection by the parasite, and no anti-T. gondii specific IgG could be detected in follow-up sera of two cases (20%), indicating false IgM initial positive results. Only 4 (40%) women showed simultaneous IgM and low avidity IgG antibodies indicating active infections. Avoidance of an over-diagnosis of acute toxoplasmosis Anti-T. gondii (IgG+IgM)/IgM prenatal screening must be supplemented by a discriminative test like IgG avidity ELISA.

Dengue: Lessons of an Outbreak.

Since 1967 there have been many outbreaks of dengue in Delhi. In the year 2015 Delhi has suffered it's one of the worst dengue outbreaks, with more than 15000 dengue confirmed cases and the highest number of deaths (60) in recent years. To determine the status of Dengue cases as compared to previous six years, the ratio of primary and secondary dengue cases and to review the effectiveness of the one test strategy (either NS1 antigen or IgM antibody) for the confirmation of Dengue. A cross-sectional study was performed in the year 2015. A total of 7177 serum samples were tested for the confirmation of suspected cases of dengue at our institute. We performed dengue NS1 antigen and dengue IgM antibody ELISA tests for the confirmation of dengue cases in acute and convalescent fever cases respectively. Hundred random samples negative for NS1 antigen were tested for IgM antibody and 100 random samples negative for IgM antibody were tested for NS1 antigen. For determination of ratio of primary and secondary dengue cases, IgG Avidity ELISA was performed on random 76 dengue positive samples. Out of 7177 samples tested, 2358 were positive either by NS1 antigen or for IgM antibody from January to December. Percentage positivity rates for IgM antibody detection and NS1 antigen detection tests were 24.8% (626) and 37.1% (1732) respectively. Out of 100 NS1 negative samples 8 were positive for IgM antibody and out of 100 IgM negative samples 6 were positive for NS1 antigen. Among the 76 samples tested for dengue IgG Avidity ELISA 52 (68.4%) were found to be of secondary dengue. Number of dengue cases is constantly rising in Delhi since 2011 and 2014. IgM antibody detection and NS1 antigen detection both the tests should be performed for each patient. Due to the increased prevalence of past infection of dengue, percentage of secondary dengue cases is also increasing in Delhi.

Role of Toxplasma gondii serology in patients with habitual abortions

ObjectiveTo study the prevalence of toxoplasmosis in pregnant females with history of habitual abortions. MethodsThe study was conducted in the Department of Microbiology, Antenatal Clinic and Gynaecology, OPD of Jawahar Lal Nehru Medical College and Hospital from February 2011 to September 2012. A total of 400 patients presented with history of multiple spontaneous abortions. On basis of history and clinical examination, 48 patients are classified as high risk group for toxoplasmosis. Serum specimens were subjected to IgG ELISA, IgM ELISA and Enzywell Toxoplasma IgG avidity ELISA test. ResultsMost of the patients were in the age group of 20-30 years. About 78.2% of females were from rural background. History of contact with cats was present in 56.5% of patients. About 91.3% of patients were non-vegetarian. Maximum number of patients with positive results in all the three serological tests had 2 spontaneous abortions. The prevalence of toxoplasmosis in pregnant females with history of habitual abortions were found to be 47.9%. IgM was positive in 35.4% while 16.6% of the patients had low IgG avidity antibodies indicating acute infection. ConclusionsRegular serological screening early in pregnancy will allow recognition of maternal infection and prompt treatment.

Seroepidemiology of Toxoplasmosis among People Having Close Contact with Animals.

A cross-sectional study was conducted to determine the seroepidemiology of Toxoplasma infection and its risk association among people having close contact with animals. A total of 312 blood samples were collected from veterinary personnel (veterinarian, technicians, and students) and pet owners from veterinary clinics and hospitals in the area of Klang Valley, Malaysia. About 4 cc of blood samples drawn from agreed participants were processed for measurement of anti-Toxoplasma IgG and IgM antibodies as well as avidity test of Toxoplasma IgG by ELISA I, II, and III kits. Meanwhile, the demographic profiles and possible risk factors of these participants were also recorded in the standardized data collection sheets. Overall seroprevalence of toxoplasmosis was observed in 62 (19.9%) participants being 7 (18.4%) in veterinarians, 15 (33.3%) in veterinary technicians, 29 (14.9%) in veterinary students, and 11 (31.4%) in pet owners. Of 19.9% Toxoplasma seropositive samples, 18.3% was positive for IgG antibody, 1.0% for IgM antibody, and 0.6% for both IgG and IgM antibodies. Of three different IgG avidity ELISA kits, ELISA III showed high avidity in all five seropositive samples (IgM and IgG/IgM antibodies) indicating chronic Toxoplasma infection which is consistent with no evidence of clinical toxoplasmosis diagnosed during the time of this study. Univariate analysis showed that age group, gender, study population, gardening, task performance, and working duration were significantly associated with Toxoplasma seropositivity. Further analysis by multivariate analysis using logistic regression showed that age group of ≥30 years old (OR = 0.34, 95% CI = 0.18–0.63, p = 0.001) and working or study duration of >10 years having close contact with animals (OR = 5.07, 95% CI = 1.80–14.24, p = 0.002) were identified as significant risks for Toxoplasma infection. Based on the results obtained, a comprehensive Toxoplasma screening and health surveillance program on toxoplasmosis should be implemented among people having close contact with animals in general and confirmed Toxoplasma seronegative individuals in particular to prevent seroconversion.

Diagnosis of Toxoplasmic Lymphadenopathy: Comparison of serology and histology

Introduction : Toxoplasma gondii is a zoonotic protozoan that infects most species of birds and mammals. It can cause neurologic or ocular disease with wide ranging manifestations. Human infection is usually asymptomatic, but some may develop fever, lymphadenopathy, malaise, chills, sweats, headaches or myalgia. Presence of lymphadenopathy is considered as a positive sign of toxoplasmosis. This is confirmed by biopsy of the inflamed lymph node. Objective : The present study was carried out to determine the reliability of the histological findings in the diagnosis of toxoplasmosis. Methods : Biopsy reports of seventeen patients who presented with lymphadenopathy were studied. ELISA was carried out in serum samples of the 17 individuals. Toxoplasma IgG avidity ELISA was performed to identify the phase of infection (acute/chronic). PCR was performed to confirm the results from ELISA. Results : Six cases were found to be seropositive. However all 17 cases had a positive biopsy report. PCR was performed to detect bands specific to T. gondii but they were absent. This result was supported by Toxoplasma IgG avidity testing which revealed that all six positive samples had high avidity thus suggesting that the infection was chronic. Conclusion : The study revealed that histological findings produced 64.7 % (11/17) false positive results. Chronic infection does not have the organism in the blood stream resulting in the absence of DNA for PCR amplification which in turn explains the absence of bands specific for T. gondi. Thus it is recommended that serology (ELISA) be used to diagnose toxoplasmosis.

IgG avidity antibodies against Toxoplasma gondii in high risk females of reproductive age group in India.

Toxoplasma gondii is an obligate intracellular protozoan that is distributed worldwide. Recently, several tests for avidity of Toxoplasma IgG antibodies have been introduced to help discriminate between recently acquired and distant infections. The study was conducted in Jawaharlal Nehru Medical College and Hospital, India from February 2011 to September 2012. Serum specimens were subjected to Toxoplasma IgM ELISA and IgG avidity ELISA test. Out of 48 patients with abortions, 17 (35.4%) were positive for IgM ELISA, and 8 (16.6%) had low IgG avidity antibodies. Out of 48 patients with other obstetric problems, 23 (47.9%) were positive for IgM ELISA, and 17 (35.4%) had low IgG avidity antibodies. Combining both groups on avidity test, only 25 of 40 (62.5%) IgM-positive women had low-avidity IgG antibodies suggesting a recent T. gondii infection in these women. More importantly, 15 (37.5%) of the IgM-positive women had high-avidity antibodies suggesting that the infection was acquired before gestation The relation of IgM seropositivity with the following risk factors was not found to be statistically significant; contact with cats (0.13), non-vegetarian food habits (0.05), and low socio-economic status (0.49). While, for IgG avidity ELISA, only contact with cats (0.01) was significantly associated with seropositivity. All other risk factors have P-values of >0.05 (not significant). IgG avidity test when used in combination with IgM test was a valuable assay for diagnosis of ongoing or recently acquired T. gondii infection in India.

Comparison of two assays in the diagnosis of toxoplasmosis: immunological and molecular

Serological tests for Toxoplasma gondii are inadequate because antibody production either fails or is significantly delayed. This study in eastern Iraq investigated the IgG-avidity ELISA test for detecting recent T. gondii infections among pregnant women and compared immunological methods and PCR as molecular assays in the diagnosis of T. gondii. Serums samples were taken from 130 pregnant women at risk of toxoplasmosis and a control group of 25 women with normal pregnancy. Of 50 IgM- and/or IgG-positive samples, only 15 showed low IgG-avidity antibodies. PCR was performed on 25 selected samples. Toxoplasma DNA was detected in 15/15 IgM-positive with low IgG-avidity and 1/3 IgM-positive with high IgG-avidity. None of the IgM-negative with high IgG-avidity showed any Toxoplasma DNA. ELISA IgG-avidity when used in combination with ELISA IgG/IgM is a valuable assay for the exclusion of ongoing or recently acquired T. gondii infection in pregnant women.

Diagnosis of antigenic markers of acute toxoplasmosis by IgG avidity immunoblotting

To perform IgG avidity immunoblotting assay for detection of acute toxoplasmosis, 100 serum samples were collected from Tehran, Iran. The presence of Toxoplasma-specific IgG and IgM antibodies were checked by commercial Trinity kit. Samples were categorized in acute and chronic phases of Toxoplasma gondii infection according to IgG avidity ELISA. IgG avidity immunoblotting was performed, and antigenic bands with molecular weights of 22, 25, 28, 30, 32, 42, 44, 49, 55, 60, 66, 69, 88, 106, 130 and 157 kDa were recognized as low avidity markers. The most prevalent antigen for low avidity was p22. It is concluded that IgG avidity immunoblotting could distinguish acute and chronic phases of T. gondii infection.

IgG Avidity ELISA Test for Diagnosis of Acute Toxoplasmosis in Humans

Serum samples, 100 in the total number, were collected from different laboratories in Tehran, Iran and tested for anti-Toxoplasma specific IgG and IgM antibodies using indirect immunofluorescent antibody test (IFAT). Using the IgG (chronic) and IgM (acute) positive samples, the IgG avidity test was performed by ELISA in duplicate rows of 96-well microtiter plates. One row was washed with 6 M urea and the other with PBS (pH 7.2), then the avidity index (AI) was calculated. Sixteen out of 18 (88.9%) sera with acute toxoplasmosis showed low avidity levels (AI≤50), and 76 out of 82 (92.7%) sera in chronic phase of infection showed high avidity index (AI>60). Six sera had borderline ranges of AI. The results showed that the IgG avidity test by ELISA could distinguish the acute and chronic stages of toxoplasmosis in humans.

IgG avidity in differential serodiagnosis of human strongyloidiasis active infection

IgG avidity assays have been developed for several parasitic diseases although there are no researches focused in strongyloidiasis diagnosis. Definitive diagnosis of strongyloidiasis is based on the presence of Strongyloides larvae in stool, but majority of cases involve low and irregular larval output. While limitations of serological assays for strongyloidiasis are well known, characteristics of persons who are misdiagnosed based on negative coproparasitological tests have been little explored. The aim of the present study was to evaluate the use of IgG avidity to detect patients with active strongyloidiasis and to characterize sources of disagreement between serology and coproparasitology. A total of 80 serum samples was analyzed, 40 from patients with Strongyloides larvae in stool (G1) and 40 from individuals with negative coproparasitology, but positive serology (G2). Serum samples were analyzed in an indirect IgG avidity ELISA using urea 6M in serial double dilutions from 1:80 to 1:2560. Avidity index (AI) was calculated to each serum dilution and analyzed as screening AI (serum dilution of 1:160) or mean AI of different serum dilutions that had a positive result. Statistical analyzes were performed by Mann–Whitney's (U) and Fisher's exact tests. At screening dilution, median of AI was 68% in G1 and 88% in G2 (P<0.0001), whereas median of mean AI in G1 was 72% and in G2 94% (P<0.0001), but there was no significant differences between both AI in each patient group. A cut off value established at AI of 75% demonstrated a significant difference between groups, with G1 sera showing AI<75% and G2 sera with AI>75% (P<0.0001). In conclusion, IgG avidity assays may distinguish active infection with Strongyloides stercoralis from suspect or serologically false positive cases.

Frequency ofToxoplasma gondiiinfection in sheep from a tropical zone of Mexico and temporal analysis of the humoral response changes

An indirect ELISA and an immunoblot were standardized to detect anti-Toxoplasma gondii antibodies in sheep, and were compared with a commercial ELISA as reference. Sensitivity was 92.0 and 96.0%, and specificity 88.0 and 75.0%, respectively. Then the serum samples of 103 sheep on a ranch located in the Eastern region of Mexico were analysed. A frequency ranging from 77 to 84% was observed, with a heterogeneous pattern among the animals by immunoblotting. Ten months later 56 sheep were sampled and tested again. Six animals became negative while 1 case was negative the first time and strongly positive 10 months later. Considering this to be a new case we calculated an incidence rate of 2.1% (CI 95% 0.6-4.8%). IgG avidity ELISA was performed on 36 positive samples, 33 being of high-avidity at both times; slight increases in 2 samples and conservation of low-avidity in 1 sheep were also observed. Higher prevalence rates of toxoplasmosis in a moist warm compared to a cold atmosphere was attributed to the long viability of T. gondii oocysts. This may explain the high frequency of T. gondii in this region, which apparently has favourable climatic conditions for the transmission of this protozoan, besides the presence of both domestic and wild cats.

Seroprevalence and Toxoplasma gondii-IgG avidity in sheep from Lajes, Brazil

Sheep are important domestic animals in the Northeast region of Brazil due to their minimal rearing and maintenance costs, and to their production of both meat and milk. In animals, Toxoplasma gondii infection results in significant reproductive and economic losses. The epidemiology of toxoplasmosis in sheep in the Northeast of Brazil has been little studied; particularly in Rio Grande do Norte State. Sera from 102 sheep intended for consumption in Lajes were subjected to the Toxoplasma-ELISA test to detect anti- T. gondii specific IgG confirming a past infection. Of the total tested, 30 (29.41%) sera were positive for IgG with an increasing number of positive animals with advancing age. We used IgG avidity ELISA in 30 positive samples and observed that 6 (20%) had low avidity antibodies and 24 (80%) had high avidity antibodies. Epidemiological studies are required in order to identify sources of infection for these hosts as well as their impact on animal breeding in the region and risk of transmission to humans.

Neospora caninum IgG avidity tests: An interlaboratory comparison

Avidity tests can be used to discriminate between cattle that are acutely and chronically infected with the intracellular parasite Neospora caninum. The aim of this study was to compare the IgG avidity ELISA tests being used in four European laboratories. A coded panel of 200 bovine sera from well documented naturally and experimentally N. caninum infected animals were analysed at the participating laboratories by their respective assay systems and laboratory protocols. Comparing the numeric test results, the concordance correlation coefficients were between 0.479 and 0.776. The laboratories categorize the avidity results into the classes “low” and “high” which are considered indicative of recent and chronic infection, respectively. Three laboratories also use an “intermediate” class. When the categorized data were analysed by Kappa statistics there was moderate to substantial agreements between the laboratories. There was an overall better agreement for dichotomized results than when an intermediate class was also used. Taken together, this first ring test for N. caninum IgG avidity assays showed a moderate agreement between the assays used by the different laboratories to estimate the IgG avidity. Our experience suggests that avidity tests are sometimes less robust than conventional ELISAs. Therefore, it is essential that they are carefully standardised and their performance continuously evaluated.

Application of the Neospora caninum IgG avidity ELISA in assessment of chronic reproductive losses after an outbreak of neosporosis in a herd of beef cattle.

Point-source infections are most likely the cause for Neospora caninum-induced abortion outbreaks in cattle, whereas an increased annual abortion rate may be a consequence of vertical transmission. The aims of the present study were to examine the reproductive effects of neosporosis in a beef herd for 3 years, after a point-source outbreak and to use IgG avidity serology to examine the chronicity of infections and patterns of transmission. During the study, 76-78% of animals were seropositive for N. caninum. The pregnancy rate varied from 88% to 94%, without any reduction in the pregnancy rate of seropositive cows compared with seronegative cows. The annual abortion rate was 2.5-5.5%, and all but 1 abortion occurred in seropositive dams. The efficiency of vertical transmission was estimated to be 85%. Several calves, born to seronegative dams, were seropositive at 6-13 months of age, indicating a 22% mean annual rate of horizontal transmission. The mean avidity in seropositive cows increased from 30 during the initial outbreak to 74 after 3 years. The mode of IgG avidity was 21-40 during the initial abortion outbreak, 41-60 after 1 year, and 61-80 after 2 and 3 years. The results reveal high annual rates of both vertical and horizontal transmission of N. caninum in a herd of beef cows and provide further validation on the ability of the N. caninum IgG avidity ELISA to accurately assess the chronicity of infection.

Assessment of antibody avidity in aborting cattle by a somatic Neospora caninum tachyzoite antigen IgG avidity ELISA.

A Neospora caninum IgG avidity ELISA was carried out on the basis of a somatic N. caninum tachyzoite antigen. The test was validated using experimentally infected calves, where a clear maturation of the IgG avidity over time could be demonstrated. At a maximum of 82 days after infection (d.p.i.), all animals showed antibody avidities ranging above 35%, and respective sera were thus defined as highly avid. Sera of 103 naturally infected seropositive cows with abortion (N. caninum association was provided by a N. caninum PCR-positivity of the fetus in 40 cases) and 139 seropositive animals without abortion history were concurrently examined. Significantly lower avidities were observed in aborting cows when compared to animals without abortion problems (P<0.01). While the avidity of sera collected before abortion remained practically constant until abortion, a significant increase of avidity could be observed in samples collected weeks to months after abortion (P<0.01). The avidities of non-aborting animals from farms with or without abortion problems did not differ significantly with time and were mainly located in the high avidity area. These data indicate that low avidities are not necessarily linked to recent N. caninum infection but can also be an indicator for increased abortion risk in cattle.