Exploration on the Detection Results of 2019 Novel Coronavirus Antibodies in the Real World

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has spread rapidly to 185 regions and countries around the world with more than 2.8 million confirmed infections and 203,044 deaths. Respiratory diseases caused by SARS‐CoV‐2 are serious threats to human health.ObjectivesTo develop a rapid detection kit for new coronavirus antibodies and use it to study the dynamic changes in antibodies in clinically confirmed SARS‐CoV‐2‐infected patients.MethodsThe SARS‐CoV‐2 IgM/IgG antibody test kit (colloidal gold method) was developed. Serum SARS‐CoV‐2 IgM and IgG antibodies were tested in SARS‐CoV‐2‐ and non‐SARS‐CoV‐2‐infected persons, respectively.Results and conclusionThe sensitivities of the SARS‐CoV‐2 IgM/IgG antibody test kit (colloidal gold method) were 50%, 70%, 92.5% and 97.5% after 1–3 days, 4–6 days, 7–9 days and >9 days of admission, respectively, and the specificities of the IgM, IgG and IgM + IgG antibodies were all 100%. Using the SARS‐CoV‐2 IgM/IgG antibody test kit (colloidal gold method), the positive rates of SARS‐CoV‐2 IgM and IgG antibodies increased from 50% to 92.5% after 1–3 days, 4–6 days and 7–9 days of admission, which showed an increasing trend. The titers of the SARS‐CoV‐2 IgM and IgG antibodies in the positive specimens increased with the length of admission.

False negative RT-PCR and false positive antibody tests–Concern and solutions in the diagnosis of COVID-19

BackgroundThis study attempts to explore the influencing factors and solutions of the colloidal gold method for novel coronavirus (2019-nCoV)-specific IgM/IgG antibody detection, summarize the clinical experience and perfect the examination process, improving the application value of antibody detection in COVID-19 diagnosis.MethodsA total of 13,329 peripheral whole blood/plasma/serum samples were obtained for COVID-19 screening from children who visited the Children's Hospital of the Capital Institute of Pediatrics outpatient clinic from April 22, 2020, to November 30, 2020. The colloidal gold method was adopted for 2019-nCoV-specific IgM/IgG antibody detection. The virus nucleic acid test results, clinical records, and serum protein fingerprint results of antibody-positive patients were collected.ResultsAll samples were examined using the colloidal gold method with two 2019-nCoV-specific IgM/IgG antibody detection kits. Four patients were tested single antibody-positive using both kits. The details were as follows: two cases of IgM ( +) and IgG (-) using plasma and serum separately, two cases of IgM (-) and IgG ( +) using serum and whole blood. The protein fingerprinting results and nucleic acid tests of 2019-nCoV antibodies were negative in the 4 cases. Considering the epidemiological history, clinical manifestations, and test results, these 4 children were ruled out for 2019-nCoV infection.ConclusionsWhen the colloidal gold method was used to detect 2019-nCoV-specific IgM/IgG antibodies, it was important to ascertain the test results as precisely as possible. Specimen type and patient history may interfere with the diagnosis.

The detection data of IgM and IgG antibodies in 169 patients with coronavirus disease‐2019 (COVID‐19) were analyzed to evaluate differences in clinical performance between the colloidal gold method and chemiluminescence method. In this study, chemiluminescence detection of IgM antibody showed a positive conversion earlier (about 1‐2 days earlier), positive conversion rates higher in different stages of disease, and a trend of declining positive rate later than colloidal gold method. For IgG antibody, the chemiluminescence method showed a positive conversion earlier and the positive rate climbing more quickly than the colloidal gold method. No obvious negative‐converting tendency of IgG detection was observed within 35 days after the onset of disease. Although colloidal gold method is generally less sensitive than chemiluminescence method, it shows advantages of shorter turn‐around time, more simple procedure, and no special equipment required. The two methodologies can be chosen according to different laboratory conditions.A reasonable understanding of the performance of reagents with different methodologies can help in clinical disease diagnosis effectively and assist in the diagnosis of the progression of COVID‐19, for which the dynamic changes of antibody will provide reliable evidence.

Objective Compare the detection result of blood samples of severe fever with thrombocytopenia syndrome (SFTS) patients using different detection techniques, and observe the dynamic characteristics of the virus specific RNA, IgM antibody and IgG antibody, to provide theoretical basis for selection of diagnostic methods of disease. Methods Acute phase serum of suspected SFTS cases and convalescent serum samples of lab-confirmed cases were collected. Real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the virus specific RNA, IgM antibody and IgG antibody. The detection results of different methods, the relationship between positive results and the acquisition time, and the dynamic characteristics of viral nucleic acid and antibodies were analyzed. Results A total of 87 serum samples of the suspected SFTS patients were collected, the positive rate of virus specific RNA, IgM antibody and IgG antibody were 53.41%, 31.03% and 3.41%, respectively. Among 55 confirmed cases of SFTS, the consistent rate of virus specific RNA and IgM antibody detection methods was 36.36%, and the difference between the two methods was significant (χ2=6.82, P=0.009), kappa=-0.257. The sampling intervals of RNA positive samples were all within 12 days, of which the positive detection rate was highest after 7-9 days, and the difference was statistically significant (χ2=10.35, P=0.016). In 34 SFTS convalescent serum samples, all the nucleic acid tests were negative, the positive rate of IgM antibody was 41.18%, which was not significantly different from the acute phase serum samples (P=1.00). The positive rate of IgG antibody was 94.12%, which was significantly higher than that of acute IgG antibody (0%). The dynamic characteristics of IgM and IgG antibody showed that IgM antibody could be detected on the second day after onset, the latest detection time was 74 days after onset, and the highest absorbance value and antibody detection rate occurred in 30-60 days. The earliest detection time of IgG antibody was 12 days after onset, and the last detection time was 100 days.The detection rate of IgG antibody and absorbance value increased rapidly after 30 days, and maintained in a high level. The detection rate of IgG antibody was 100% in 30-60 days. Conclusions Blood samples taken from SFTS suspected patients within two weeks of onset may be prioritized for detection of viral nucleic acids using Real-time fluorescence PCR or for detection of IgM antibodies by ELISA. Although IgM antibody can be detected 2 days after the onset, the peak appeared much later, so the negative result can’t rule out the diagnosis. IgG antibody has a high seroconversion rate in convalescent samples, and can be used as an auxiliary tool for disease diagnosis. Key words: Severe fever with thrombocytopenia syndrome; SFTS virus; Nucleic acids; Antibodies; Real-time fluorescent quantitative PCR; Enzyme-linked immunosorbent assay

BackgroundCOVID‐19 has become a global pandemic, and close contacts and asymptomatic patients are worthy of attention.MethodsA total of 1844 people in close contacts with 76 COVID‐19 patients were investigated, and nasopharyngeal swabs and venous blood were collected for centralized medical quarantine observation. Real‐time fluorescence was used to detect SARS‐CoV‐2 nucleic acid in nasopharyngeal swabs of all close contacts, and the colloidal gold method was used to detect serum‐specific antibodies. Levels of IgM‐ and IgG‐specific antibodies were detected quantitatively through chemiluminescence from the first nucleic acid turned negative date (0 week) and on weekly intervals of ≤1 week, 1–2 weeks, 2–3 weeks, 3–4 weeks, 4–5 weeks, 5–6 weeks, and 6–7 weeks.ResultsThe total positive rate of the colloidal gold method (88.5%, 23/26) was significantly higher (χ2 = 59.182, p < 0.001) than that of the healthy control group (2.0%, 1/50). There was significant difference in IgG concentration at different time points (0–7 weeks) after negative nucleic acid conversion (χ2 = 14.034, p = 0.029). Serum IgG levels were significantly higher at weekly time points of 4–5 weeks (Z = −2.399, p = 0.016), 5–6 weeks (Z = −2.049, p = 0.040), and 6–7 weeks (Z = −2.197, p = 0.028) compared with 1–2 weeks after negative nucleic acid conversion. However, there was no significant difference (χ2 = 4.936, p = 0.552) in IgM concentration between time points tested (0–7 weeks) after negative nucleic acid conversion. The positive rates of IgM and IgG in asymptomatic patients (χ2 = 84.660, p < 0.001) were significantly higher than those in the healthy control group (χ2 = 9.201, p = 0.002) within 7 weeks of negative nucleic acid conversion.ConclusionsThe IgG concentration in asymptomatic cases remained at a high level after nucleic acid turned negative. Nucleic acid detection combined with IgM and IgG antibody detection is an effective way to screen asymptomatic infections.

To explore the application of the colloidal gold method and chemiluminescence method in detecting gonadotropin (Gn) in morning urine for assessing pubertal development status in children. A total of 132 children diagnosed with central precocious puberty (CPP), early and fast puberty (EFP), and premature thelarche (PT) at Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from November 2021 to December 2022 were included, along with 685 healthy children who underwent routine health examinations at the hospital's pediatric health care department during the same period. All 132 patients underwent a gonadotropin-releasing hormone (GnRH) stimulation test. Both patients and healthy children had their urinary Gn levels measured using the colloidal gold method and chemiluminescence method, including levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The correlation between serum Gn and urinary Gn detected by the two methods, as well as the correlation between Tanner stages of healthy children and urinary Gn, was analyzed. Urine Gn levels detected by both the colloidal gold method and chemiluminescence method showed a positive correlation with serum LH baseline values, LH peak values, baseline LH/FSH ratios, and peak LH/FSH ratios (P<0.05). In healthy children, urinary LH levels detected by the chemiluminescence method gradually increased from Tanner stage Ⅰ to Ⅳ (P<0.05), while urinary FSH levels were lower in Tanner stage I than in stages Ⅱ, Ⅲ, and IV (P<0.05). Urinary LH levels detected by the colloidal gold method were lower in Tanner stage I compared to stages Ⅱ, Ⅲ, and IV, with the highest levels observed in Tanner stage Ⅳ (P<0.05). Additionally, urinary FSH levels in Tanner stage Ⅲ were higher than in stages Ⅰ and Ⅱ (P<0.05). The area under the receiver operating characteristic curve for evaluating Tanner stages I and II in healthy children using urinary LH and FSH levels by the chemiluminescence method and urinary LH levels by the colloidal gold method were 0.730, 0.699, and 0.783, respectively. The colloidal gold method and chemiluminescence method for detecting Gn in morning urine show good correlation with serum Gn levels. As a non-invasive and convenient detection method, the colloidal gold method can serve as a useful tool for screening the onset of pubertal development in children.

False-positive treponemal syphilis serology linked to EBV-related heterophile antibodies: Insights from a multi-platform diagnostic

IntroductionTo evaluate the performance of oral fluid (OF) for quantitative detection of SARS-CoV-2 nucleic acid and IgG antibody, and explore its application value, we compared OF with traditional samples using multiple detection methods.MethodsReal-time PCR (RT-PCR) and digital PCR (dPCR) were used to detect SARS-CoV-2 nucleic acid in 213 paired OF and throat swab samples from COVID-19 cases. Chemiluminescence was applied to detect IgG antibody in paired OF and serum samples, while colloidal gold method was used specifically for OF.ResultsRT-PCR results showed that the positivity rates of OF and throat swabs were 80.75% (172/213) and 88.73% (189/213) respectively (p < 0.001). Using throat swabs as the reference, the sensitivity of OF for nucleic acid detection by RT-PCR was 87.83% (172/189). For 108 paired samples analyzed by dPCR, the positivity rate of OF was 86.11% (93/108), slightly higher than that by RT-PCR (85.19%, 92/108). Notably, in some cases, the viral load in OF exceeded that in throat swabs, accounting for 38.89% (N gene) to 41.67% (ORF1ab gene) of the tested samples.ConclusionAs a non-invasive, convenient, safe, and self-collectible biological sample, OF shows high consistency in detection efficacy compared with traditional throat swabs and serum. It thus holds important application value for the diagnosis, monitoring, and epidemic prevention and control of COVID-19.

To compare the performance of a new chemiluminescence method with that of the traditional colloidal gold method for cryptococcal antigen (CrAg) detection. Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. Cryptococcal antigen (CrAg) testing from serum and cerebrospinal fluid (CSF) has been regarded as the gold standard for early diagnosis. In this study, a total of 140 samples (92 serum and 48 cerebrospinal fluid samples) from 140 patients with suspected cryptococcosis collected between January 2022 and September 2023 at Zhejiang Provincial People's Hospital were tested via a fully automated chemiluminescent immunoassay analyser (SuperFlex) from Suzhou Xinbo and a cryptococcal antigen detection kit (colloidal gold method) from the IMMY Company of the United States. According to the diagnostic criteria for cryptococcosis, 55 of the 140 suspected patients were diagnosed with cryptococcosis (39.3%), including 47 with pulmonary cryptococcosis (PC) and 8 with cryptococcal meningitis (CM). The degree of agreement between chemiluminescence and the colloidal gold method was analysed via Cohen's kappa coefficient, which was 0.970 (P<0.01). The sensitivities of the chemiluminescence and colloidal gold methods were 98.2% and 96.4%, respectively, and their specificities were 100% and 98.8%, respectively. The area under the receiver operating characteristic (ROC) curve were 0.996 for chemiluminescence and 0.9759 for the colloidal gold method. The area under the curve (AUC) of the two methods did not differ significantly (P=0.086). For the detection of CrAg, the new chemiluminescence method is highly consistent with the traditional colloidal gold method and has higher sensitivity and specificity for the diagnosis of cryptococcosis.

Nucleic acid detection plays an important role in environmental monitoring, food safety, pathogen detection, genetic disease detection and so on. At present, nucleic acid detection methods mainly include nucleic acid molecular hybridization and polymerase chain reaction. Nucleic acid detection of Corona Virus Disease 2019 has become an indispensable part of our daily life. With the establishment of new detection methods, nucleic acid detection is developing towards single molecule sensitivity and single base specificity. Colloidal gold method and magnetic particle chemiluminescence method have been widely used in the autonomous detection of COVID-19，while both approaches have their limitations. There is a more efficient and specific approach: CRISPR-Cas13 system

We aimed to explore the duration of IgM antibodies against Mycoplasma pneumoniae. Data from two children who consistently tested positive for M. pneumoniae IgM antibodies were retrospectively analyzed. Moreover, we examined the etiological data and drug use of these cases. Serologic testing using the colloidal gold method, direct chemiluminescence technique, and specific immune agglutination test were utilized. Quantitative PCR was used to detect M. pneumoniae in bronchoalveolar lavage fluid and antigen tests and nucleic acid detection were conducted for other respiratory pathogens. The serological positivity of M. pneumoniae IgM antibody persisted for nearly ten months in one child and more than fifteen months in the other child. Furthermore, the persistently positive M. pneumoniae IgM antibody tests led to the inappropriate use of macrolides during multiple hospitalizations. IgM antibodies against M. pneumoniae may remain positive for an extended duration. Therefore, a positive Mycoplasma pneumoniae IgM test does not necessarily indicate the presence of an acute infection.

Real-time polymerase chain reaction (RT-PCR) of virus nucleic acid test (NAT) has become the standard method to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, there are still many limitations, especially the problem of the high false negative rate. Therefore, the aim of this study was to investigate the positive rate of SARS-CoV-2 NAT and evaluate the diagnostic performance of SARS-CoV-2 IgM and IgG antibody detection in novel coronavirus infection. A total of 10309 suspected or high-risk cases of infection with SARS-CoV-2 in Wuhan Hubei, China, were tested for virus NAT by RT-PCR. Among those cases, 762 COVID-19 patients and 143 patients with non-COVID-19 who were tested for SARS-CoV-2 IgM and IgG during the NAT period were screened. The difference between the two test methods was analyzed using the chi-square test. The positive rate of 10309 cases was about 36% (95% CI: 33.39%-39.67%). SARS-CoV-2 was present in various types of specimens, and alveolar lavage fluid had the highest positive rate [52.38% (95% CI: 31.02-73.74)]. The clinical sensitivity of serum SARS-CoV-2 IgM and IgG was 77.17% (588/762) and 94.88% (723/762), respectively, and the clinical specificity was 93.71% (134/143) and 90.21% (129/143). The area under the curve (AUC) of SARS-CoV-2 IgG and combination of IgG with IgM were equally larger than IgM [0.973 (95% CI: 0.964-0.983) vs 0.930 (95% CI: 0.910-0.949)]. IgG antibody had the highest specificity [100.0% (95% CI: 100.00%-100.00%)] and sensitivity [94.0% (95% CI: 92.45%-95.55%)] when detected alone or in combination with IgM antibody. The total coincidence rate of SARS-CoV-2 antibodies detection and SARS-CoV-2 NAT for the diagnosis of SARS-CoV-2 infection was 92.04% (833/905). Among the 34 SARS-CoV-2 NAT-negative patients with clinical symptoms and CT imaging features, 29 (85.29%) patients were positive for SARS-CoV-2 IgM, and 31 (91.76%) were positive for IgG. SARS-CoV-2 NAT should be considered for many types of specimens, and the combined test of SARS-CoV-2 IgM and IgG can make up for the problem of missed NAT in COVID-19 patients.

Background Heterophile antibody (HAb) interferences in immunoassays can cause falsely elevated hCG concentrations leading to incorrect diagnosis and treatments options. When results are not consistent with the clinical findings, hCG HAb interference investigation may be requested by the physician. A retrospective evaluation of the frequency of HAb interference was performed among cases of physician-requested investigations and the effectiveness of commercially available blocking reagents to detect HAb interference in two immunoassay systems was evaluated. Methods One hundred and thirteen physician requests for hCG HAb investigation from 2008 to 2017 were reviewed. The primary method used to measure hCG was the Beckman Coulter Access Total βhCG (2008-2010) and the Roche Elecsys HCG+β (2014-2017). HAb investigation included measurement by two immunoassays before and after treatment of samples with heterophile blocking reagents and serial dilution studies. Results Five cases of HAb and HAb-like interference were identified. The interference frequency was 6.7% for the Beckman assay and 2.9% for the Roche assay. The presence of HAb was detected using heterophile blocking reagents and an alternative method in three cases. The other two cases were detected due to discrepant results with an alternative method and non-linear serial dilutions (HAb-like). Conclusions HAb interference was observed in the Beckman and the Roche assays. The heterophile blocking reagents failed to detect 40% of interference cases. Blocking reagents should not solely be used for these investigations. Multiple strategies including the use of serial dilutions and using an alternative platform are critical when troubleshooting interferences in hCG immunoassays.

An automated, fully-integrated nucleic acid analyzer based on microfluidic liquid handling robot technique