Immunoglobulin G Enzyme-linked Immunosorbent Assay Research Articles

Redefining Gold Standard Testing for Diagnosing Leptospirosis: Further Evidence from a Well-Characterized, Flood-Related Outbreak in Sri Lanka.

A gap in the leptospirosis field remains the lack of well-characterized sample collections that allow for comparison of new methods to standard ones. In the context of a flood-related outbreak of leptospirosis evaluated in Anuradhapura, Sri Lanka, a specimen bank was obtained with detailed metadata accompanied by gold standard diagnostic test results. Blood samples collected on admission and 14 days later from suspected cases of leptospirosis were tested using microscopic agglutination test (MAT) (17 serovars), an in-house enzyme-linked immunosorbent assay (ELISA) using a locally obtained strain of Leptospira kirschneri as sonicated antigen, a commercially available ELISA based on sonicated Leptospira biflexa, and a quantitative polymerase chain reaction (qPCR) assay targeting the pathogenic Leptospira-specific 16S rRNA gene. Of 62 patients presenting within the first 2 days of illness, 31 had confirmed leptospirosis based either on paired-sample MAT or qPCR. During the acute phase, qPCR was most sensitive, detecting 74% of definitively diagnosed cases; immunoglobulin G (IgG) ELISA (in-house), IgG ELISA (commercial), and MAT had sensitivities of 35.5%, 12.0%, and 22.6%, respectively, in detecting definitively diagnosed cases using acute phase serum. Of 40 patients with paired sera, 10 were qPCR positive. Of these, five samples were negative by paired-sample MAT. Of the 11 MAT-positive samples, only five were detected using qPCR confirming that both tests are needed for maximal sensitivity. Regional leptospiral serovar-specific IgG ELISA was superior to MAT. Knowing the regionally dominant serovars improves serological sensitivity in the analysis of acute specimens by ELISA, but qPCR was most sensitive in this patient population.

Dendritic cell vaccination enhances antiangiogenesis induced by endostatin in rat glioma.

It has been verified that dendritic cell (DC) vaccination can improve the prognosis of malignant glioma. However, recent evidence suggests the problems with DC vaccines lies, at least in part, with the cancers ability to induce an immunosupressive response that suppresses any vaccine-mediated active immunity. Our previous studies indicate that subcutaneous vaccine can restrain the cancer cells implanted in the brain, but the effect is limited on vascularized tumor in the brain. Furthermore, vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule (VCAM) play an important role in immunoevasion. To identify the effects of DC vaccination on antiangiogenesis induced by endostatin in rat glioma. Rat basal ganglia glioma model was constructed and authenticated. The concentration of immunoglobulin G (IgG) was detected using rat IgG enzyme-linked immunosorbent assay (ELISA) kit. CD8+ T cell infiltration was measured by immunofluorescence. The expression of VEGF, VCAM, and intercellular adhesion molecule 1 (ICAM-1) tested by real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. The expression of VEGF and apoptosis in rat glioma tissues is tested by immunohistochemical staining. Two group comparisons were analyzed by a two-tailed Student's t test. Multiple group comparisons were analyzed by one-way analysis of variance (ANOVA). P values less than 0.05 were considered statistically significant. The combination of DC vaccination and antiangionesis inhibited the rats with malignant glioma by stimulating immune response, supressing the expression of angiogenesis-related proteins VEGF, VCAM, and ICAM-1. In addition, the combination therapy inhibited glioma stem-cell-like cells. DC vaccination enhances antiangiogenesis induced by endostatin in rat glioma.

Abstract A08: Distribution, cutoff, and functional significance of a potential biomarker for Imprime PGG, an experimental cancer immunotherapeutic, in a healthy subject population

Abstract Imprime PGG (Imprime) is a yeast-derived beta-1,3/1,6 glucan that binds complement receptor 3 (CR3) on innate immune cells and enables these cells to exert anti-tumor activity against tumor cells that have been opsonized by iC3b following targeting by anti-tumor antibodies. Following incubation with whole blood (WB) from healthy subjects, Imprime was shown to bind to neutrophils and monocytes via CR3 and modulate complement receptor expression, activation marker expression, and interleukin-8 (IL-8) production. These effects, however, differed widely among subjects. It was subsequently observed that Imprime-induced functional activities occurred predominantly in individuals with higher levels of endogenous immunoglobulin G (IgG) or IgM anti-beta-glucan antibodies (ABA) and that ABA are a prerequisite for opsonizing Imprime to allow its binding to CR3. A small pilot study (n=32) with healthy subjects using prototype enzyme-linked immunosorbent assays (ELISAs) to measure IgG and IgM ABA suggested correlations between ABA levels, Imprime binding to neutrophils in WB, and consequent induction of functional responses. The objectives of this study were to further optimize and qualify the prototype ELISAs and, in a larger cohort of healthy subjects (n=143), to confirm the significance of elevated ABA levels with respect to binding of Imprime to neutrophils and monocytes and Imprime–induced functional changes. Qualification of the IgG and IgM ABA ELISAs yielded good inter-assay precision (coefficients of variation <10%), linearity (recovery 97-112%), and dynamic range (2.5 logs). Incubation of WB samples with Imprime resulted in high binding to neutrophils (>5% of cells) in samples from 52% of the subjects. Samples from these same subjects also exhibited high Imprime binding to monocytes. ABA levels in the high binding individuals were significantly greater than those in the low binding individuals (p<0.0001 for IgG and p=0.01 for IgM). IgG ABA levels were more highly correlated with Imprime binding to neutrophils and monocytes (r=0.64 and r=0.61, respectively) than IgM ABA levels (r=0.22 and r=0.33, respectively). There was no correlation of ABA levels with age, gender, or total serum immunoglobulin concentration. Receiver operator characteristic (ROC) curve analysis comparing sensitivity and specificity of ABA levels vs. Imprime binding to neutrophils yielded an area under the curve (AUC) of 0.89 for IgG and 0.70 for IgM and generated several options for cutoffs. At one proposed ABA cutoff set at 95% specificity for binding, 36% of subjects had IgG ABA levels ≥ the cutoff, 20% had IgM ABA levels ≥ the cutoff, and 46% had levels ≥ either cutoff. The sensitivity to detect high Imprime binding to neutrophils was 64% for IgG and 33% for IgM, with a combined sensitivity of 80%. Similar correlations were seen with Imprime binding to monocytes. In subsets of the healthy population in which functional evaluations were also performed following in vitro exposure to Imprime, IgG ABA levels at or above vs. below the cutoff correlated with higher C4a (n=32; p=0.0003), C5a (n=32; p<0.0001), and SC5b-9 (n=32; p<0.0001) production, increased production of IL-8 (n=129; p<0.0001), and increased neutrophil and monocyte CR3 expression (n=40; p=0.006 and n=37; p=0.009 respectively). Data presented here, using the further optimized ELISAs for measurement of IgG and IgM ABA in WB samples from a larger healthy population, confirm that serum ABA levels correlate with in vitro neutrophil as well as monocyte binding and response to Imprime. Furthermore, ABA cutoffs at high specificity could identify the majority of healthy individuals whose cells demonstrated this response. The use of ABA as a clinical biomarker for Imprime response in cancer patients is under investigation. Citation Format: Diane McMurray, Ben Harrison, Katie Ertelt, Richard Walsh, Lindsay Wurst, Steven Leonardo, Nadine Ottoson, Adria Bykowski Jonas, Xiaohong Qiu, Nandita Bose, Peter Maimonis. Distribution, cutoff, and functional significance of a potential biomarker for Imprime PGG, an experimental cancer immunotherapeutic, in a healthy subject population. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A08.

Detection of Chikungunya Virus in Nepal.

Chikungunya virus (CHIKV) is an emerging alphaviral disease and a public health problem in South Asia including Nepal in recent years. In this study, sera were collected from patients presenting with fever, headache, muscular pain, fatigue, and joint pain of both upper and lower extremities. A total of 169 serum samples were tested for CHIKV and dengue virus (DENV) by using Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibody using enzyme-linked immunosorbent assay (ELISA) method during August to November 2013. Results showed that 3.6% and 27.8% samples were positive for CHIKV and DENV IgM positive, respectively. Similarly, results of IgG showed 3.0% samples were positive for CHIKV IgG and 29.0% were for DENV IgG positive. Further, a 50% focal reduction neutralization test (FRNT50) was performed to confirm the presence of CHIKV, which demonstrated that 8.9% of CHIKV IgM and/or IgG ELISA positive possessed neutralizing anti-CHIK antibodies. To our knowledge, this is the first report in which the presence of CHIKV is confirmed in Nepalese patients by FRNT50. Basic scientists and clinicians need to consider CHIKV as a differential diagnosis in febrile Nepalese patients, and policy makers should consider appropriate surveillance and actions for control strategies.

A new Toxoplasma gondii chimeric antigen containing fragments of SAG2, GRA1, and ROP1 proteins-impact of immunodominant sequences size on its diagnostic usefulness.

This study presents the first evaluation of new Toxoplasma gondii recombinant chimeric antigens containing three immunodominant regions of SAG2, GRA1, and one of two ROP1 fragments differing in length for the serodiagnosis of human toxoplasmosis. The recombinant chimeric antigens SAG2-GRA1-ROP1L (with large fragment of ROP1, 85–396 amino acid residues) and SAG2-GRA1-ROP1S (with a small fragment of ROP1, 85–250 amino acid residues) were obtained as fusion proteins containing His6-tags at both ends using an Escherichia coli expression system. The diagnostic utility of these chimeric antigens was determined using the enzyme-linked immunosorbent assay (ELISA) for the detection of specific anti-T. gondii immunoglobulin G (IgG). The IgG ELISA results obtained for the chimeric antigens were compared to those obtained for the use of Toxoplasma lysate antigen (TLA) and for a mixture of recombinant antigens containing rSAG2, rGRA1, and rROP1. The sensitivity of the IgG ELISA was similar for the SAG2-GRA1-ROP1L chimeric antigen (100 %), the mixture of three proteins (99.4 %) and the TLA (97.1 %), whereas the sensitivity of IgG ELISA with the SAG2-GRA1-ROP1S chimeric antigen was definitely lower, reaching 88.4 %. In conclusion, this study shows that SAG2-GRA1-ROP1L chimeric antigen can be useful for serodiagnosis of human toxoplasmosis with the use of the IgG ELISA assay. Therefore, the importance of proper selection of protein fragments for the construction of chimeric antigen with the highest reactivity in ELISA test is demonstrated.

Validation and evaluation of VapA‐specific IgG and IgG subclass enzyme‐linked immunosorbent assays (ELISAs) to identify foals with Rhodococcus equi pneumonia

Rhodococcus equi (Rhodococcus hoagii/Prescottella equi) is a common cause of foal pneumonia, but its diagnosis remains a challenge for equine veterinarians. While the VapA-specific (virulence-associated protein A) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) has low sensitivity and specificity for detecting pneumonic foals, little is known about VapA-specific IgG subclasses. To evaluate the performance of VapA-specific ELISA for IgG and its subclasses IgGa, IgGb and IgG(T) in the early diagnosis of pneumonia caused by R. equi. Assay validation followed by assessment of diagnostic performance using archived samples from animals of known status. Serum samples from exposed (n = 125) and nonexposed adult horses (n = 10) and from experimentally challenged and naturally infected foals were used for ELISA validation. Post mortem and tissue culture records of the last 24 years from the Institute for Experimental Pathology at the University of Iceland in Keldur, Iceland laboratory were evaluated to confirm the absence of R. equi cases in Iceland. The diagnostic performance of VapA-specific IgG and its subclasses was evaluated using banked serum samples from pneumonic (n = 21) and healthy foals (n = 80). To evaluate each IgG assay, a cut-off value was selected based on receiver operating characteristic curve analysis and used to calculate sensitivity and specificity. The intra- and interassay coefficients of variation were calculated for each ELISA. Using sera from Iceland, where R. equi infection has not been reported, the VapA-specific IgG ELISA differentiated exposed from nonexposed horses. When used to identify infected foals, VapA-specific IgG, IgGa and IgGb had no diagnostic value. In contrast, IgG(T) had high sensitivity and specificity. Horses from Iceland are not exposed to VapA(+) R. equi and can serve as negative controls. VapA-specific IgG subclasses, with the exception of IgG(T), are poor predictors of disease. Further investigation on the use of IgG(T) as a diagnostic tool in field conditions is needed.

Detection of Brucella immunoglobulin G among high risk group: Significance in the diagnosis and follow-up

Introduction: Human brucellosis is clinically indistinguishable from other infectious diseases such as tuberculosis, typhoid, leptospira, and malaria. Laboratory diagnosis remains a mainstay in the diagnosis of brucellosis. This study was carried out to find out the prevalence of brucellosis among high risk group by Brucella immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) testing to assess the magnitude of the problem. Materials and Methods: A total of 130 blood samples collected from high risk group, another 130 samples from general population as control group were tested for Brucella IgG ELISA using smooth-lipopolysaccharide antigen. Results: Among the 130 high risk group, 19 (15%) were positive for Brucella IgG antibody. The duration of exposure of more than 25 years was observed in 10 (53%) seropositive cases. 21 (16%) handled Brucella vaccine for animals. Of this, 13 were seropositive for Brucella IgG. Among the 130 control group, none were positive for Brucella IgG antibody. Conclusion: Since vaccine is not available for human, awareness of transmission of brucellosis, vaccination of livestock, protective measures like barrier protection, while handling animal vaccines and isolation of infected animals can reduce the prevalence.

Serosurvey of Crimean-Congo hemorrhagic fever virus in domestic animals, Gujarat, India, 2013.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral disease that causes a fatal hemorrhagic illness in humans. This disease is asymptomatic in animals. CCHF was first confirmed in a nosocomial outbreak in 2011 in Gujarat State. Another notifiable outbreak occurred in July, 2013, in Karyana Village, Amreli district, Gujarat State. Anti-CCHF virus (CCHFV) immunoglobulin G (IgG) antibodies were detected in domestic animals from the adjoining villages of the affected area, indicating a considerable amount of positivity against domestic animals. The present serosurvey was carried out to determine the prevalence of CCHFV among bovine, sheep, and goat populations from 15 districts of Gujarat State, India. A total of 1226 serum samples from domestic animals were screened for IgG antibodies using a CCHF animal IgG enzyme-linked immunosorbent assay (ELISA) kit from the Centers for Disease Control and Prevention. Antibodies were detected in all the 15 districts surveyed; with positivity of 12.09%, 41.21%, and 33.62% in bovine, sheep, and goat respectively. This necessitates the surveillance of CCHFV IgG antibodies in animals and hemorrhagic fever cases in human.

Evaluation of Different Serological Diagnostic Methods for Tick-Borne Encephalitis Virus: Enzyme-Linked Immunosorbent, Immunofluorescence, and Neutralization Assay

Tick-borne encephalitis (TBE) is a zoonotic disease, transmitted mainly by the bite of ticks. The TBE virus (TBEV) belongs to the family Flaviviridae, genus Flavivirus and is able to cause meningoencephalitis. For serological TBEV detection, the neutralization test (NT) is the most specific assay available. Different NT protocols are used in the laboratories, and until now the performance of these NTs has never been tested in an external quality assessment (EQA). In this EQA, we compared the results of eight European laboratories in detecting 17 samples (11 TBEV positive, five flavivirus cross reactive, and one negative sample) by NT. Furthermore, 14 of these EQA samples and 15 additional samples were tested in different commercial assays: 15 immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) and an immunofluorescence assay (IFA). Four laboratories showed a good NT EQA performance, whereas four laboratories had some sensitivity problems. Additionally, two of these laboratories showed a lack in specificity, misidentifying a dengue-positive sample as TBEV positive. The comparison of the commercial ELISAs revealed a high sensitivity in all assays, but as expected for IgG, the ELISAs showed a high degree of flavivirus cross reactivity. The assessment of Vienna Units in some of the ELISAs revealed deviations in the standards used by the different companies. Therefore, these standards should be revised. Generally, in this EQA, we found that reliable NT protocols are used in most of the laboratories, and the evaluation of the IgG ELISAs and the IFA showed a good agreement.

Diagnostic performance of selected commercial HEV IgM and IgG ELISAs for immunocompromised and immunocompetent patients

BackgroundHepatitis E virus (HEV) genotype 3 is recognised as an emerging pathogen in industrialised countries. The currently commercially available HEV-specific enzyme linked immunosorbent assays (ELISAs) are primarily designed for the detection of antibodies against genotypes 1 (Burma) and 2 (Mexico) and may not sensitively detect HEV genotypes 3 or 4. ObjectivesThis study aimed to evaluate the analytical and clinical performances of eight commercially available HEV serum antibody immunoglobulin M (IgM)- and immunoglobulin G (IgG)-specific ELISAs for genotype 1 and 3 HEV infections in a clinical setting and to study the antibody responses against HEV of immunocompromised versus immunocompetent patient groups. Study designAnalytical performance and diagnostic sensitivity and specificity were assessed using well-defined reference samples and samples from patients with polymerase chain reaction (PCR)-confirmed HEV infection (n=88) and a specificity panel (n=98). ResultsLimiting dilutions indicated that the highest analytical sensitivity in head-to-head comparison was measured for the Mikrogen_new IgG assay. Taking the serum working dilutions of each assay into account, the Wantai IgG assay was the most sensitive assay. Receiver operator curve (ROC) analysis showed area under the curve (AUC) values of 0.943, 0.964, 0.969, 0.971, 0.974 and 0.994 for the DSI, Mikrogen_old, MP Diagnostics, Mikrogen_new, Wantai and DiaPro anti-HEV IgM assays, respectively. The highest specificity of currently available assays was found for the IgM Wantai assay (>99%). If anti-HEV IgM and IgG results from each supplier were combined, DSI and Wantai assays were able to detect the highest number of (passed) HEV infections. ConclusionsOur study showed that current commercial HEV ELISAs could be used to diagnose HEV genotype 3 infection adequately in a clinical setting.

Recombinant envelope protein-based enzyme immunoassay for IgG antibodies is comparable to neutralization tests for epidemiological studies of dengue infection

Dengue virus (DENV) is the most prevalent arbovirus in the world, found mainly in tropical regions. As clinical manifestations present frequently as nonspecific febrile illness, laboratory diagnosis is essential to confirm DENV infections and for epidemiological studies. Recombinant envelope (E) antigens of four serotypes of DENV were used to develop an immunoglobulin G enzyme-linked immunosorbent assay (IgG-ELISA). To evaluate the IgG-ELISA, a panel of serum samples that had been tested previously by a plaque reduction neutralization test (PRNT) was investigated for the presence of anti-E antibodies against the four DENV serotypes. IgG-ELISA was found to have a sensitivity (91%) and specificity (98%) at a receiver-operating characteristic (ROC) optimized cutoff and demonstrated high performance as well as good indexes. A concordance of 97% was achieved between both assays, and only 21/704 (3%) samples were not concordant. The results of the present study demonstrate a moderate correlation between neutralizing antibody titers and IgG-ELISA values. These findings indicate that the recombinant protein-based IgG-ELISA is a suitable method for routine serodiagnosis, monitoring and seroepidemiological studies of DENV infections.

Comparison of Two Enzyme Immunoassays for DetectingMycoplasma pneumoniae

Objective: Mycoplasma pneumoniae is an important etiologic agent in primary atypical pneumonia. This study examined the correlation between results of several serologic tests commonly used for the diagnosis of M. pneumoniae infection. Methods: We compared results of 2 enzyme immunoassay (EIA) kits. Sera from 221 patients with M. pneumoniae were tested using the Vircell M. pneumoniae enzyme-linked immunosorbent assay (ELISA) and the ZEUS Scientific Mycoplasma IgG and IgM ELISA kits. Results: The sera from 221 samples were tested for M. pneumoniae-specific immunoglobulin M (IgM), and 161 of those sera were tested for immunoglobulin G (IgG). Overall IgM–positive rates were 13% for the Vircell and 28% for the ZEUS kits, and corresponding IgG-positive rates were 21% and 38%, respectively. The overall agreement between results of the Vircell and ZEUS ELISAs were 77% (95% confidence interval [CI], 71-82%) for IgM and 74% (66-80%) for IgG. Conclusion: Although the results of IgM EIAs showed relatively high positivity in the early acute phase, the results of EIAs undertaken for the detection of M. pneumoniae infection showed only moderate concordance. Therefore, more accurate and reliable diagnostic methods are needed for this condition.

Cytomegalovirus Neutralization by Hyperimmune and Standard Intravenous Immunoglobulin Preparations

Cytomegalovirus (CMV) remains one of the most important pathogens after transplantation, potentially leading to CMV disease, allograft dysfunction, acute, and chronic rejection and opportunistic infections. Immunoglobulin G (IgG) preparations with high antibody titers against CMV are a valuable adjunctive prevention and treatment option for clinicians and apart from standard intravenous immunoglobulin (IVIG), CMV hyperimmune preparations are available. The CMV antibody titer of these preparations is typically determined by Enzyme-linked immunosorbent assay (ELISA), also used for the selection of high titer plasma donors for the production of the CMV Hyperimmune product. However, CMV ELISA titers do not necessarily correlate with CMV antibody function which is determined by virus neutralization tests. CMV antibody titers were determined by both ELISA and virus neutralization assay and the IgG subclass distribution was compared between a CMV hyperimmune licensed in Europe and standard IVIG preparations. Although the expected high CMV IgG ELISA antibody titers were confirmed for three lots of a CMV hyperimmune preparation, the functionally more relevant CMV neutralizing antibody titers were significantly higher for 31 lots of standard IVIG preparations. Moreover, considerably lower IgG3 levels were found for the CMV hyperimmune preparation compared with standard IVIG preparations. The higher functional CMV neutralization titers of standard IVIG preparations and the better availability of these preparations, suggest that these products could be a valuable alternative to the CMV hyperimmune preparation.

Antibody Response After a Single Dose of an AS03-Adjuvanted Split-Virion Influenza A (H1N1) Vaccine in Heart Transplant Recipients

Influenza A (H1N1) has emerged as a considerable threat for recipients of organ transplants. Vaccination against the novel influenza A (H1N1) virus has generally been advocated. There is limited experience with AS03-adjuvanted A/H1N1 pandemic influenza vaccines in immunosuppressed patients. We conducted an observational, nonrandomized single-center study to assess antibody response and vaccine-related adverse effects in 47 heart transplant recipients (44 men; age, 56±13 years). The AS03-adjuvanted, inactivated split-virion A/California/7/2009 H1N1v pandemic vaccine was administered. Antibody titers were measured using hemagglutination inhibition; immunoglobulin G (IgG) response was assessed using a new pandemic influenza A IgG enzyme-linked immunosorbent assay (ELISA) test kit and compared with hemagglutination-inhibition titers. Adverse effects of vaccination were assessed by a questionnaire. Postvaccination antibody titers of greater than or equal to 1:40 were found in only 15 patients, corresponding to a seroprotection rate of 32% (95% confidence interval, 19%-47%). Sensitivity, specificity, positive predictive value, and negative predictive value of ELISA testing were 80.0%, 68.8%, 54.5%, and 88.0%, respectively. Age, time posttransplantation, and immunosuppressive regimen did not impact antibody response. Vaccination was well tolerated. Single-dose administration of an AS03-adjuvanted vaccine against the novel influenza A (H1N1) virus did not elicit seroprotective antibody concentrations in a substantial proportion of heart transplant recipients; the new pandemic influenza A IgG ELISA test kit proved to be of limited clinical use.

Immunogenicity and Efficacy in Hemodialysis Patients of an AS03 A-Adjuvanted Vaccine for 2009 Pandemic Influenza A(H1N1): A Nonrandomized Trial

Hemodialysis patients have a reduced response to vaccinations because of uremia-related immune dysfunction. To increase the immunogenicity of vaccines, antigens can be formulated with adjuvants. The new tocopherol-containing adjuvant system AS03(A) has not been tested yet in patients with end-stage renal disease. Nonrandomized trial. 291 hemodialysis patients from 3 dialysis units co-operating with the Department of Nephrology at the University Hospital Heidelberg, Germany: 169 patients were vaccinated using either 1 (64 patients) or 2 doses (105 patients); 123 patients refused the vaccination and served as controls. Intramuscular immunization with 3.75 μg of an inactivated split-virion A/California/7/2009 H1N1v pandemic vaccine adjuvanted with AS03(A) in a single- or double-dose regimen. A pandemic influenza A immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) response >11 AU (arbitrary units) was defined as a positive response. Quantitative antibody testing using the pandemic influenza A IgG ELISA. Antibody titers were tested 3 months after vaccination and compared with nonimmunized dialysis patients. After vaccination against 2009 pandemic influenza A(H1N1), 41 of 64 (64.1%) patients with 1 vaccination and 93 of 105 (88.6%) with 2 vaccinations showed a protective immune response compared with 43 of 123 (34.9%) unvaccinated patients (P < 0.001). Logistic regression analysis confirmed vaccination dose as an independent factor for response to pandemic H1N1 vaccination. No episode of pandemic H1N1 illness occurred in any group within the study period of 6 months after vaccination. No serious adverse events occurred, and local symptoms ranged from mild to moderate in 143 of 169 (84.6%) patients. Nonrandomized assignment; use of nontreated patients as controls; no comparison to nonadjuvanted vaccines; dose variation in the intervention group. Pandemic H1N1 vaccine adjuvanted with AS03(A) is immunogenic, effective, and safe in hemodialysis patients.

An Outbreak of Lethal Toxoplasmosis in Pigs in the Gansu Province of China

In October 2004, a swine farm in Jinchang, Gansu Province, China, experienced an outbreak of toxoplasmosis. Most of the affected pigs had a rectal temperature greater than 40 degrees C and gradually lost their appetite. Morbidity reached 57%, and mortality was approximately 2%. Analysis of blood samples from affected pigs using hemagglutination inhibition (HI) assay, immunoglobulin G-enzyme-linked immunosorbent assay (IgG-ELISA), and IgM-ELISA tests showed high titers of anti-Toxoplasma gondii antibody. Tachyzoites of T. gondii were found in body fluids of mice inoculated intraperitoneally with ground samples from the heart, liver, spleen, and brain of 2 sick pigs. In addition, the inoculation of 5 pigs with T. gondii tachyzoites caused death in 2 of the pigs. The origin of this outbreak was concluded to be food-borne T. gondii infection.

Reactivation of latent Toxoplasma gondii in immunocompromised cancer patients

Active Toxoplasma gondii infection in cancer patients undergoing anticancer chemotherapy was evaluated using both enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) techniques. Ninety-nine cancer patients were studied. Sixty seven of those patients were under cancer chemotherapy treatment, 41 (61.2%) of which were found to be ELISA immunoglobulin g (IgG) positive and five (7.5%) were PCR positive. On the other hand, none of the 32 untreated cancer patients was found to be PCR positive although 22 (68.7%) of them were ELISA IgG positive. These findings suggest the possibility that treatment with immunosuppressive agents predisposed cancer patients to reactivation of T. gondii infection.

Detection of Caprine Herpesvirus 1–Specific Antibodies in Goat Sera Using an Enzyme-Linked Immunosorbent Assay and Serum Neutralization Test

An enzyme-linked immunosorbent assay (ELISA) was developed to detect immunoglobulin G (IgG) antibodies directed to whole Caprine herpesvirus 1 (CpHV-1). Sera from 248 goats were obtained from CpHV-1-free and CpHV-1-infected flocks and were subjected to both IgG ELISA and serum neutralization (SN) assays, with the latter considered the gold standard for the diagnosis of CpHV-1 infection. In flocks where CpHV-1 infection was detected, 57 sera were negative by the SN and the ELISA tests and 97 sera were positive with both tests. Thus, although based on biologically different principles, the ELISA was as sensitive as the SN assay in detecting seropositive animals and could be efficiently used as a faster and less expensive alternative to the SN test for the screening of many samples.

Utilizing rabbit immunoglobulin G protein for mark-capture studies on the desert subterranean termite, Heterotermes aureus (Snyder)

Mark-capture dispersal studies were conducted to investigate the feasibility of marking the southwestern desert subterranean termite, Heterotermes aureus (Snyder) with rabbit immunoglobulin G (IgG). In turn, short-range dispersal patterns of H. aureus were measured across a 20-m diameter desert landscape at three distinct field locations. Each location consisted of 51 termite feeding stations containing corrugated cardboard. The central feeding station (CFS) at each location was impregnated with rabbit IgG. A circular grid was then constructed around each CFS that consisted of 50 additional unmarked cardboard feeding stations strategically placed around the CFS at distances of 1.5, 2.0, 4.0, 7.0 or 10.0 m. Termites self-marked with rabbit IgG by feeding on the marked bait. The CFS and the 50 peripheral feeding stations were sampled for marked termites twice at each location 17-65 days after the marked bait was placed at the CFS to determine the spatial dispersal patterns of H. aureus within each research grid. Termites that self marked by feeding on rabbit IgG marked bait were detected by an anti-rabbit IgG enzyme-linked immunosorbent assay (ELISA). Generally, the CFSs contained the highest frequency of marked termites with 28.0% of the individuals assayed from the CFSs containing rabbit IgG. Over the course of the study, 39 of the unmarked peripheral feeding stations contained at least one marked termite. Of the termites assayed from the peripheral stations (n = 2,955), 124 or 4.2% of the individuals contained the mark. The average distance traveled by the marked termites collected at the peripheral feeding stations was 5.7 ± 3.3 m from the CFSs. We also examined single nucleotide polymorphisms (SNPs) from termites collected at each field site. Data indicated that each field site were genetically distinct and therefore non-related termites. We discuss the advantages and limitations of marking termites with rabbit IgG for dispersal studies.

West Nile Virus Seroprevalence in Blood Donors from Central Anatolia, Turkey

West Nile virus (WNV) is a reemerging flavivirus that has displayed a drastic change in epidemiology in the last decade. Data on WNV activity in Turkey are currently limited. This study investigated WNV exposure in blood donors from Central Anatolia, Turkey. A total of 2516 sera, collected from blood donors at four major branches of the Turkish Red Crescent Middle Anatolia Regional Blood Center, were evaluated by a commercial WNV immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA). Positive and borderline samples were investigated further by a WNV IgG indirect immunofluorescence test (IIFT), IgG ELISAs for tick-borne encephalitis virus and dengue virus, an IgG IIFT for yellow fever virus, and a multi-Flavivirus biochip IgG IIFT. WNV antibody specificity and titer values were determined by plaque reduction neutralization assay. IgG avidity and IgM were determined for confirmed samples. IgM-positive samples were also evaluated by a real-time reverse transcription polymerase chain reaction assay. Twenty-five samples (25/2516; 0.99%) were found reactive in the WNV ELISA/IIFT assays, and 14 could be confirmed by the plaque reduction neutralization assay (14/2516; 0.56%). All IgGs were of high avidity, and four samples (4/14; 28.6%), which were negative for viral RNA, were IgM positive. Although samples with neutralizing WNV IgGs had strong fluorescence intensity in IIFTs, no correlation between antibody titer values and IIFT intensity or quantitative ELISA results could be found. Three WNV nonreactive samples were positive in the dengue IgG ELISA test; one of these also displayed positive results for dengue virus in the mosaic biochip IIFT and reactivity in yellow fever virus IIFT. WNV exposure is confirmed in 0.56% of the tested healthy blood donors in Central Anatolia, with evidence for dengue/yellow fever and/or other flaviviral infections. This study is the first to document WNV exposure in individuals from Konya, Yozgat, and Sivas provinces in Central Anatolia, and it also establishes viral activity in Ankara, the capital of Turkey.