Expression, Purification, and Application of SARS-CoV-2 Nucleocapsid Protein for Serological Detection of IgG and IgM Antibodies.

Objectives: The clinical utility of complementary tests for brucellosis are not clear in many situation. This study aimed to evaluate value of these tests for brucellosis in an endemic area in Turkey. Materials and methods: This study was performed at Çanakkale General Hospital in 2009. In a retrospective approach, records of the patients who evaluated for brucellosis were collected. During the study period, 236 people (131 symptomatic and 105 non-symptomatic) were evaluated for diagnosis of brucellosis. All of the samples from these patients were tested for Brucella antibody seropositivity by RB slide agglutination, standard serum agglutination, Brucella Coombs, BrucellaCapt, and ELISA IgG and IgM tests. Results: In total, 49 symptomatic patients were hospitalized and blood cultures were obtained. Brucella spp. were isolated from nine of them (18.4%).The BrucellaCapt test was found to be the most sensitive for Brucella (74.0%) and close behind it was the Coombs test (72.5%). The sensitivity for the RB test was 48.1%. The ELISA IgG test was found more sensitive for brucellosis than the ELISA IgM test was (65.6% and 49.6%, respectively). All examined tests were found about 100% specific for brucellosis but the RB test was found less specific than the others were (96.1%) Positive predictive value for all tests was about 1 but negative predictive values were only valuable for the Coombs and Brucella Capt test (0.744 and 0.755, respectively). The other serological tests were around and below 0.50, which was weak for negative results. Conclusions: The ELISA IgG and IgM tests were no superior to the other tests. By assessment of receiver operating characteristics (ROC) analysis, the Brucella Coombs and BrucellaCapt tests were found to be the most valuable tests for serological diagnosis of brucellosis in endemic areas. The seronegative tests in the symptomatic patients should be evaluated and repeated in short time.

Helicobacter pylori is main causative agent of acute gastritis and peptic ulcer disease. In certain population, this infection leads to gastric cancers viz., adenocarcinoma, and mucosal-associated lymphoid tissue (MALT) lymphoma. The objective of this study was to comparatively evaluate invasive (RUT) and non-invasive (IgM and IgG ELISA) methods for detection of H. pylori infection among patients visiting a tertiary care hospital. A total of 285 dyspeptic patients undergoing endoscopic examination were included in this study. From each patient one biopsy specimen and serum samples were collected. Biopsy specimen was subjected to RUT and IgM & IgG ELISA tests were performed using serum samples. H. pylori was detected in 127 (44.6%) and 126 (44.2%) cases by RUT and IgM ELISA, respectively. H. pylori were detected in 85 (29.8%) samples by IgG ELISA. Based on the combination of RUT and IgM ELISA test, total 128 (44.9%) patients were positive for H. pylori infection. Most of the positive cases belonged to 21-40 years age group (60 of 128) followed by 41-60 years age group (31 0f 128). All the three diagnostic methods viz., RUT, IgM ELISA and IgG ELISA used in this study showed a greater prevalence of H. pylori infection in female gender compared to male gender. In this study, sensitivity of both RUT and IgM ELISA was similar across different age groups and gender. The advantage of IgM ELISA over RUT is that it does not require endoscopy. Therefore, IgM ELISA could be considered as safe and an alternative method for detection of this pathogen.

I evaluated performance of the Mycoplasma pneumoniae-specific IgM antibody rapid detection test (ImmunoCard Mycoplasma, IC, Meridian, USA) and compared it to the particle agglutination (PA) test and ELISA tests (Mycoplasma pneumoniae IgG, IgA, IgM ELISA medac, Medac Diagnostika, Germany). Serum samples numbering 112 were obtained from 70 pediatric patients (< 16 years old) with M. pneumoniae infection diagnosed by a PA test (four-fold or greater rise by paired serum samples or > or = 1:640 by a single serum sample). Of these, 82 samples (73.2%) were positive in IC and 91 (81.3%) positive in ELISA IgM tests. Specifically, for samples obtained within 7 days following the onset of fever, 6 of the 14 positive in the ELISA IgM test were negative in IC and 4 of the 18 samples negative in the ELISA IgM test were positive in IC. I ascribed this difference to the difference in antigens used in each test. In the analysis of sequential serum samples from 2 patients with M. pneumoniae pneumonia, IC was still positive in 248- and 527-day samples for which a PA test and the ELISA IgM and IgG tests indicated no acute infection. Nine (36.0%) of 25 serum samples obtained from apparently healthy adult volunteers were positive in IC. Of the 9 IC-positive cases, ELISA tests suggested possible recent infection at most in 3 cases, while the remaining 6 cases had no evidence of acute infection. In conclusion, although IC is sufficiently sensitive as a rapid screening test for detecting M. pneumoniae-specific IgM antibody, a positive result in the test does not always indicate acute infection by this organism. To ensure accurate diagnosis of M. pneumoniae infection, paired serum samples are thus required for conventional methodologies.

It has been well recognized throughout the world that Helicobacter pylori is the main cause of gastric ulcer and stomach carcinoma. Laboratory diagnosis of H. pylori infection is made by invasive and non-invasive methods. Invasive methods require endoscopy which is uncomfortable an unacceptable by the most patients. Therefore, non-invasive methods particularly serological tests are easier and comfortable for patients. A total of 86 patients with ages ranging from 18-77 years old (43 males and 43 females) who were referred to the Duhok Hepatology &amp; Gastroenterology center/Azadi Teaching Hospital for endoscopic examination from June to October, 2013 were enrolled in the study. From each patient 5 ml of blood was collected under a septic condition and sera were separated for serology. Data from each participant were recorded in a special questionnaire form after consent agreed upon on ethical and scientific committee of the Azadi hospital. The efficacy of three different ELISA tests (IgG, IgM, IgA) was assessed taking culture as a gold standard method. pylori was found in 70.93%, 30.23% and 5.81% by ELISA IgG, IgA and IgM tests respectively. The highest percentage (70.93%) of H. pylori positive cases were found by ELISA IgG and the lowest percentage (5.81%) was recorded in ELISA IgM. The study found a significant relationship between age groups and H. pylori positive cases by ELISA IgG in which positive cases increased with the increase of ages.&#x0D; No statistical correlation was found between the sex, smoking status and residency of studied patients with H. pylori infection. A significant correlation was found between patients without endoscopic lesions and positive cases for H. pylori by ELISA IgG in which 75.67% of patients without endoscopic lesions were positive for anti-H. pylori antibodies compared to 41.66% in patients with endoscopic lesions.&#x0D; The efficacy of IgG, IgM and IgA tests was calculated compared with golden standard tests and ELISA IgG characterized by the highest values of sensitivity (75.75%), negative predictive value (36.00%) and accuracy (86.00%) but with lowest specificity (45.00%) and similar positive predictive value (81.96%) with IgM and IgA. The lowest sensitivity was 5.97% and accuracy 25.56% with ELISA Ig M. From the results of the present study we can conclude that among ELISA tests, anti-H. pylori IgG is a reliable serological test for the diagnosis of H. pylori infection.

Chikungunya virus is an Arbo virus belonging to the family Togaviridae. In urban areas, antropophilic Aedes aegypti and Aedes albopictus mosquitoes are vectors for virus transmission to human population. Chikungunya virus has attracted the professional and scientifi c public attention in 2013 causing a massive outbreak on the American continent. In Europe, autochthonous transmissions of Chikungunya virus infections have beenrecorded in Italy in 2007 as well as in France in 2010 and 2014. Usutu virus is a RNA virus from the family Flaviviridae. Th e virus circulates in a transmission cycle between wild birds and Culex mosquitoes. The virus has been detected in numerous bird species across Europe. Manifestations recorded in humans include meningoencephalitis and skin rash. First human cases in Europe were recorded in immunocompromised individuals in Italy in 2009. Spirochete Borrelia burgdorferi sensu lato is transmitted to humans by ticks and causes Lyme disease, a multisystemic disease with dermatological, neurological, cardiological or articular manifestations. Ninety three persons interviewed about risk factors for vector-borne infections were examined. The examination was performed using commercial ELISA IgG for Chikungunya and Usutu virus and ELISA IgM and IgG test for Borrelia burgdorferi in line with manufacturer’s instructions (Euroimmun, Germany). Out of 93 examined individuals Usutu virus specific IgG antibodies were identified in 7.5% (7/93) persons. Th e results of ELISA IgG test for Chikungunya virus were negative in the majority of tested samples, whereas 7.5% (7/93) of samples revealed borderline result. In 9.7% (9/93) participants, antibodies against Borrelia burgdorferi were detected only by ELISA IgM test. Recent infection with Borrelia burgdorferi was confi rmed in 2.15% (2/93) individuals, whereas IgG antibodies against Borrelia burgdorferi were detected in only one participant. Th e obtained results indicated that Usutu virus is active in the territory of South Bačka District contrary to Chikungunya virus as well as that Borrelia burgdorferi is an important pathogen in the investigated region.

Objective: Brucellosis is one of the most common zoonotic diseases in our country and the world. This study investigated the seroprevalence of brucellosis in individuals who were engaged in animal husbandry in Erzurum region, were over 18 years of age, or did not have any clinical complaints of brucellosis. Materials and Methods: Our study was conducted on 271 volunteers engaged in livestock farming in 20 districts of Erzurum province between 2020 and 2021. Brucella antibodies were investigated in blood samples using ELISA IgG, ELISA IgM, Rose Bengal, and Standard tube agglutination tests. A questionnaire was administered to the participants to investigate the risk factors of brucellosis. Results: The mean age of the participants was 43.6 ± 15.7.2 years (min:18, max:90), and there were 137 male (50.6%) and 134 female (49.4%) particpants. Seropositivity was detected with ELISA IgG in 40 of 271 participants (14.8%). Seropositivity was detected in the ELISA IgM test in 2 of these 40 participants (0.7%). Seropositivity was found in 46 participants (17%) in the Rose Bengal and 29 individuals in the STA test. According to the serology results, ELISA IgG positivity was higher in male participants compared to female participants (ratio=1.6). Among pet owners, ELISA IgG seropositivity was found higher than the participants who did not have pets (ratio=1.9). In the evaluation based on educational status, the rate of seropositivity was slightly higher in the participants who were illiterate and had a poor level of education. Conclusion: In our study, it was determined that the prevalence rate obtained by ELISA IgG was consistent with previous studies conducted in the region. No significant relationship was observed between the serology results of the participants and their gender and educational status. On the other hand, the positivity of ELISA IgG results was higher among the participants who had pets, and there was a significant relationship at the margin.

Toxoplasma specific IgM antibodies; the common serologic marker in diagnosis of acute toxoplasmosis has its own limitations. Confirmatory testing with other markers, introduced as a complementary tool in distinguish acute and chronic infections is unusual in Iran. In the present study, we investigated the correlation between the results of IgM ELISA, IgA ELISA, and IgG avidity tests in the diagnosis of toxoplasmosis to demonstrate the necessity of confirmatory testing in serodiagnosis of infection in the country. A total of 107 positive Toxoplasma IgG and IgM sera were obtained from patients referred to private laboratories and stored at -20 °C for futures use. Serologic tests were set up in duplicate to analyze the serum levels of IgG, IgM, IgA, and IgG avidity antibodies using commercial ELISA kits. The results were presented as semi quantitative for IgG, IgM and IgA ELISA, and Relative Avidity Index in percentage for IgG avidity test. Pearson's correlation coefficient (rp) was applied to analyze the data. Of 107 serum samples, T. gondii specific IgM and IgA antibodies were positive in 67.3% and 53.3%, respectively. Besides, 29.9% of the sera displayed low avidity for IgG antibodies. The rp was - 0.572 (P<0.01) between the IgG avidity and IgM ELISA, - 0.364 between the IgG avidity and IgA ELISA (P<0.01), and 0.564 between the IgM and IgA ELISA (P<0.01). The study strongly highlights the necessity of confirmatory testing in differential diagnosis of acute and chronic toxoplasmosis in Iran.

SummaryThe diagnostic value of Brucella ELISA IgG and IgM has been evaluated in patients with brucellosis. Serum samples and blood cultures were collected from 83 patients with brucellosis. The sera were tested by Brucella ELISA for Brucella IgM and IgG antibodies. All 44 controls were negative for IgG and IgM. Brucella melitensis was isolated from blood cultures of 30/83 (36.1%) patients. Among the 30 bacteremic patients, 24 (80%) had an increased IgM titer of ≥200. Of the 53 non-bacteremic patients, 41 had IgM titer ≥200, while 22 had IgG titer of ≥1,600. The ELISA IgM and IgG tests achieved a specificity and sensitivity of 100% and 96% respectively, while the positive and negative predictive values were 100% and 94% respectively. The Brucella ELISA is a reliable and sensitive test in the diagnosis of brucellosis. The test is rapid, easy to perform and can be automated.

The aim of this work was to ascertain the usefulness of a new commercially-available single-assay chemiluminescence test (CHT) for the diagnosis of human tularemia (Tularaemia VIRCLIA IgG + IgM monotest, Vircell, Santa Fe, Granada, Spain). A total of 773 sera from 773 patients including 364 initial sera from patients with diagnosed tularemia, patients with suspected tularemia not confirmed (100), healthy people (152), patients with serology positive to Brucella (97), patients diagnosed with other infectious diseases (30), and patients diagnosed with autoimmune diseases (30) were included. All sera were tested by CHT, "in-house" microagglutination test (MAT), immunochromatographic test (ICT) (Virapid Tularaemia, Vircell, Santa Fe Granada, Spain), and "in-house" ELISA IgG, and ELISA IgM. Of the total initial sera, 334 (sensitivity 91.8%) were positive in the CHT, 332 (sensitivity 91.2%) in the MAT, 330 (sensitivity 90.7%) in the ICT, and 328 (sensitivity 90.1%) in the ELISA IgG and ELISA IgM tests. The specificity of the CHT was 96.7%; of the MAT, 100%; of the ICT, 98.7%; and of the ELISA IgG and ELISA IgM, 97.4%. In the group of patients with serology positive to Brucella, at least 12.4% of sera were positive in tularemia tests (12.4% in ELISA IgM, 13.4% in MAT, 14.4% in ICT, and 15.5% in CHT and ELISA IgG). In conclusion, CHT presents a sensitivity and specificity in early diagnosis of human tularemia, similar to MAT, ICT, and ELISA IgG and ELISA IgM. Its single assay design allows lower costs, especially in areas of low endemicity or inter-epidemic periods.

West Nile virus (WNV) is arbovirus distributed all around the world. In humans, 80% of infection cases are asymptomatic. In 20% of infected people, a febrile self-limiting illness is reported. WNV has the potential for fatal neuroinvasive disease. In 1% of cases, the infection may result in neuroinvasive disease with permanent neurological consequences or death outcome. Neurological forms may vary presenting with encephalitis, meningitis, meningoencephalitis or acute flaccid paralysis. Outbreaks with neurological forms of WNV infection were recorded in different areas of Greece, Italy, Romania, Hungary and Serbia. During the period from 2013 to 2016, 114 samples of cerebrospinal fluid and 107 serum samples were taken from 114 patients suspected of WNV neuroinvasive disease (WNND). The presence of specific anti-WNV IgM and IgG antibodies in cerebrospinal fluid (CSF) and sera samples were tested by WNV IgM and IgG ELISA (Euroimmun, Germany). In addition, 48 samples of CSF or/and serum of people with suspected WNV infection were examined by commercial molecular tests - real time RT-PCR (WNV Real-TM, Sacace biotechnologies, Italy). The IgM antibodies against WNV were present in 25.4% (29/114) of CSF samples, and in 31.8% (34/107) of serum samples tested from 114 patients suspected of WNND. The IgG antibodies against WNV were detected in 3.5% (4/114) of CSF samples, and in 11.2% (12/107) of serum samples. The WNV RNA was detected by real time RT-PCR test in 7 out of 48 (14.6%) CSF or/and serum samples. In this study, detection of IgM antibodies in CSF is more frequent than detection of WNV RNA in CSF or serum samples. WNV RNA detection in CSF is confirmatory diagnostic test but has limited utility in the diagnosis of WNV neuroinvasive disease due to low viremia level at the time of clinical presentation of the disease. The limitations in the use of ELISA IgM test are linked to cross - reactivity among flaviviruses and long persistence of IgM antibodies in the serum and CSF.

Background and study aim : ELISA can determine specific antibody classes against brucella, It is a sensitive, simple and rapid test, thus help to study the pattern of the brucellosis. The aim of this article is to study the pattern of the brucellosis in Menoufyia governorate. . Patients and Methods: Sera from 150 individuals from confirmed brucellosis cases and 25 healthy individuals were tested for presence of IgG and IgM antibodies by ELISA assay. Culture positivity for brucellosis was used as the reference standard for diagnosis. Results: Serum IgG and IgM for brucellosis by ELISA test had increased values in confirmed brucella cases, ELISA IgM was highly specific (100%) in all groups and sensitive (96%) in acute brucellosis, (100%) in subacute brucellosis and (64%) in chronic brucellosis. While, ELISA IgG specificity in all groups was (80%) and the sensitivity in acute brucellosis was (88%), in subacute and chronic brucellosis was (100%). Conclusion: ELISA IgG and IgM test for brucella is a simple and reliable test in the diagnosis of pattern of the brucellosis.

Toxoplasma gondii is a worldwide opportunistic protozoan causing life-threatening infection in immunocompromised patients, while frequently asymptomatic in immunocompetent individuals. The current study aimed to detect T. gondii; serologically and molecularly in ß. thalassemia patients and evaluate the association of infection with some hematological parameters in these patients. Blood samples were collected from 100 ß. thalassemia patients. Serological diagnosis of T. gondii using ELISA for IgG and IgM antibodies was performed. Molecular diagnosis by Real-Time (RE) PCR was performed using specifically designed primers amplifying 389bp fragments of Toxoplasma genome. 45 patients (45%) had anti-Toxoplasma IgG antibodies with no detectable IgM antibodies while both anti-Toxoplasma IgM and IgG antibodies were noticed in 10 patients (10%). IgM only antibodies were discovered in two cases (2%). The total seropositivity rate among patients was 57%. RE PCR analysis revealed Toxoplasma DNA in 20% out of 100 patients. PCR and serological examination showed slight agreement. A statistically significant relation was observed between the results of IgG and IgM ELISA and PCR for the detection of T. gondii infection among patients with ß. thalassemia. None of the studied risk factors (age, gender, contact with cats, consumption of undercooked meat) or hematological parameters (ESR, anemia degree, ferritin level, type of blood transfusion, spleen status) showed statistically significant association with Toxoplasma infection. It can be concluded that patients with thalassemia have a high risk of infection with T. gondii. RE PCR should be used as a diagnostic method in association with serology especially in immunocompromised patients to increase sensitivity.

Clinical performance of a semi-quantitative assay for SARS-CoV2 IgG and SARS-CoV2 IgM antibodies

BackgroundThe kinetics and specificity of anti-ZIKV antibodies after acute ZIKV infection is not well known, especially in areas where different flaviviruses circulate. The objective of this study was to describe the kinetics of anti-ZIKV antibodies in pregnant women in whom an acute ZIKV infection was identified during pregnancy.MethodsWithin a cohort of pregnant women living in Guadeloupe and exposed to ZIKV during the 2016 zika outbreak, we identified 65 women who presented with an acute, symptomatic PCR-confirmed ZIKV infection at various times of their pregnancy, with a known date of first Zika symptom. Anti-ZIKV neutralizing antibodies (using a Virus Neutralisation Test (VNT)) and anti-ZIKV NS1 antibodies (using IgM and IgG ELISA Euroimmun® kits) were searched for on frozen serum samples obtained from blood drawn at the time of delivery in all women and at various times between acute infection and delivery in 23 women.ResultsPatients’ mean age was 30 years and ZIKV infection had occurred during the first, second, and third trimester of pregnancy in 14 (21%), 35 (54%), and 16 (25%) women, respectively. ZIKV serology on delivery samples was positive in 65/65 (100%; one-sided 97.5% CI: 94.4%–100%) women by both VNT and IgG ELISA assays and in 5/65 (8%) women by IgM ELISA assay. In these 5 cases, median time between first symptom and sampling date was 36 days. Results of ELISA assays on the intermediate samples were as follows: IgG antibodies were negative in all 5 samples that had been drawn within 7 days of first symptom and positive in the 18 samples that had been drawn afterwards; IgM antibodies were positive in 10 of the 19 samples that had been drawn within 3 months of first symptom and negative in 2 of the 4 samples that had been drawn afterwards.ConclusionAfter acute ZIKV infection, IgG antibodies developed and remained detectable until delivery by a commercially available ELISA assay in all women tested. These antibodies were specific of ZIKV, with concomitantly positive VNT results. From these findings, the absence of ZIKV antibodies at delivery would strongly indicate the absence of infection during pregnancy.DisclosuresAll authors: No reported disclosures.

An indirect hemagglutination test (IHAT) and an ELISA test for trichinosis using as antigen a larvae soluble fraction from Trichinella spiralis was carried out for the detection of IgG, IgM and IgA specific antibodies in 113 serum samples from patients confirmed or suspected to have trichinosis by strong clinical and epidemiological evidences (Group I). The same tests were also performed on 110 serum samples corresponding to patients without strong evidences of having trichinosis (Group II). In Group I the corresponding sensitivities for RHAI, ELISA IgG, ELISA IgM, ELISA IgA were: 82.3-85.8-88.5 and 88.5% respectively. Seventeen patients were tested again a week after the first analysis (10 of them corresponded to negative ones), increasing the positivity: 23.5-100.0;35.3-100.0;41.2-100.0 and 41.2-100.0% for RHAI, ELISA IgG, ELISA IgM and ELISA IgA, respectively. Other two patients were followed-up for 5 years. IHAT and ELISA IgG remained positive, whereas ELISA IgM and ELISA IgA were constantly negative betweeen 17 and 32 months in one case, and between 48 and 60 months in the other (this last one had presented a severe clinic disease). In the group II, four patients were positive with IHAT, however only one for ELISA IgA, the latter also presented ELISA IgM near the cut off. The use of ELISA IgG, ELSIA IgM and ELISA IgA in the immunodiagnosis of trichinosis is discussed.