Humoral Immune Durability of Anti-SARS-CoV-2 Antibodies in Southern Iran: A Population-Based Survey Study

Background: To promote mitigation strategies and public health response, this study aimed to evaluate the population-based seroprevalence of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in Hormozgan province. Materials and Methods: The study was conducted within 10 districts of Hormozgan province with 1325 participants for three months by considering three-month successive follow-ups to evaluate the durability of humoral immunity. The participants completed the questionnaire, and blood samples were taken followed by immunoassay SARS-CoV-2 ELISA testing. Results: In general, 717 (54.1%) males and 596 (45.9%) females participated in this study. In phase one, 147 (11.1%) and 182 (14.7%) tested positive for immunoglobulin M (IgM) and IgG, respectively. Upon three months, 13.8% and 17.8% tested positive for IgG and at least for one of the antibodies. Based on the results, 606 (45.7%) cases reported no symptoms while 673 (50.8%) of them reflected at least one. Among 798 (60.2%) participants, the most common symptoms were headache (n = 244, 18.4%), sore throat (n = 186, 14.0%), weakness (n = 150, 11.3%), muscular pain (n = 139, 10.5%), and sputum cough (n = 134, 10.1%). The odds of the antibodies in females was 1.37 (95% CI: 1.03, 1.82, P = 0.03). In phase 2, 43 (5.3%) participants persisted positive for IgG while 559 (73%) tested negative for IgG. Finally, 20% of the suffered participants tested positive for IgG until nine months. Conclusion: Although IgG antibodies decreased in the first six months, their titers persisted stable for nine months. It seems our population has not reached a desirable level of protection. It is stressed that mass vaccination is needed to prevent future epidemic waves.

RESULTS OF THE QUESTIONNAIRE ON THE PRESENCE OF VIRAL INFECTION (COVID-19) OF A.N. SYZGANOV NSCSEMPLOYEES

По данным ВОЗ на 25 ноября 2020 г. официально зарегистрированных случаев COVID-19 60,3 миллиона человек в мире, 38,6 миллионов выздоровевших, летальный исход 1,42 миллиона. Казахстан также является одной из стран, серьезно пострадавших от COVID-19. В период с конца июня по июль 2020 г. в Казахстане произошел резкий рост заболеваемости, республика находилась в «красной» зоне из-за неутешительной эпидемиологической ситуации. Целью исследования явилась изучение эпидемиологической ситуации по COVID-19 среди сотрудников ННЦХ им. А.Н. Сызганова. Всего в исследование вошли 384 сотрудника в возрасте от 21 до 80 лет, составил 44,1 ± 0,3 года. Сотрудникам центра проводились исследования методом полимеразно - цепной реакции (ПЦР) диагностика, иммуноферментный анализ (ИФА) тестирование (на антитела к SARS-CoV-2), иммунохемилюминесцентный анализ (ИХЛА) (для выявления общих антител к SARS-CoV-2) и компьютерная томография (КТ) грудной клетки. В результате исследования всего анкетировано - 384 сотрудников, из них по анкете заболевших вирусной инфекцией - 174 человека (45,7 %). Был сделан вывод, что 76,6% персонала переболели в легкой степени Ковид - 1 COVID-199, и доказана была благополучная эпидемиологическая ситуация с выскочим сформированным коллективным иммунитетам. Рекомендуем по полученным результатам исследования, что нецелесообразно определять антитела к COVID - 19 в течения месяца, лучше определять с помощью метода ИХЛА через три месяца. According to the WHO, there are 60.3 million officially registered COVID-19 casesin the world, 38.6 million recovered, and a death rate of 1.42 millionby November 25, 2020. Kazakhstan is also one of the countries severely affected by COVID-19. In the period from the end of June to July 2020, there was a sharp increase in the incidence in Kazakhstan, moreover, Kazakhstan was in the "red" zone due to the disappointing epidemiological situation. A study of the viral infection (COVID-19) epidemiological situationamong employees of the A.N. Syzganov NSCS. The centerstaff were studied by the method of polymerase chain reaction (PCR) diagnostics, enzyme-linked immunosorbent assay (ELISA) testing (for antibodies to SARS-CoV-2), сhemiluminescent immunoassay (CLIA) (to identify common antibodies to SARS-CoV-2) and computer tomography (CT) of the chest. In total, the study included 384 employees of the NSCS aged from 21 to 80 years, average age was 44.1 ± 0.3. 81 men and 303 women were examined: 54 of them were doctors, 188 nurses, 90 junior medical staff, and the other 52 employees. In total, 384 employees were interviewed (questioned), 174 (45.7%) were infected with a viral infection according to the questionnaire. In accordance with studyresults, it can be concluded that 76.6% of NSCS employees have had a viral infection (COVID - 19) and a sufficient immune layer has formed in the team to maintain a favorable epidemiological situation in the next six months. Based on the studyresults, we recommend, that it is inappropriate to determine antibodies to COVID-19 within a month, it is better to determine using the CLIA method after three months

Proinflammatory status of oral fluid in COVID-19

At present, a search for promising ways to diagnose infection caused by SARS-CoV-2 is quite relevant. Oral fluid is not commonly used for assessment of COVID-19 risk. Its molecular profile reflects both local state of the oral cavity, and individual organs and systems, thus suggesting a reliable diagnostic platform. Systemic inflammatory response is known to play a crucial role in development of the coronavirus infection; the “cytokine storm” determines severity of the disease. The saliva-based diagnostics of clinical course in COVID-19 patients includes determination of IL-6, IL-8, C-reactive protein in oral fluid, in order to assess severity of the inflammatory process. The present study was carried out at the Department of Fundamental and Clinical Biochemistry with Laboratory Diagnostics, and Department of Pediatric Infections at the Samara State Medical University. The study involved 122 persons: 67 clinically healthy individuals comprised the control group, and the group of comparison included 55 inpatients with moderate or severe coronavirus infection (COVID-19) caused by SARS-CoV-2 virus as confirmed by PCR and/or ELISA testing. Development of the disease was accompanied by drastically increased contents of IL-6 and IL-8 in oral fluid of the patients relative to the indexes in healthy persons, i.e., several-fold for IL-6 (+ 650%) and even higher elevation of IL-8 levels (+ 26513%), as well as a 2-fold increase of C-reactive protein (+115%). When comparing the immune indexes of oral fluid in presence versus absence of respiratory insufficiency, a significant difference was found for salivary IL-6 (+173%) in the patients with grade 1-2 respiratory insufficiency as compared with patients free of respiratory disorders. Determination of these proinflammatory markers in patients with COVID-19 is of important prognostic significance when assessing development of the disease and its severity. Direct detection of their content in the oral fluid makes this method relevant, and potentially demanded for the outpatient diagnostics, being highly important during pandemics of coronavirus infection and limited medical resources. Examination of oral fluid at the pre-hospital stage is a resource-saving technology, since it does not require additional medical staff to take biomaterial, is non-invasive to the patient, and suggesting a wide range of research items, it can resolve a number of diagnostic issues, e.c., presence of specific genetic material or antibodies to SARS-CoV-2, severity of the inflammatory process and the risk of respiratory failure in the patient.

A Road Paved with Good Intentions: A Platelet Count-Based Alert to Facilitate Diagnosis of Heparin-Induced Thrombocytopenia

Background: Heparin-induced thrombocytopenia (HIT) is a rare disorder with potential to cause significant morbidity and mortality. Early identification and initiation of non-heparin anticoagulation can prevent devastating thrombotic events. However, over-testing is common and can lead to result misinterpretation, unnecessary heparin avoidance, and increased cost. When there is concern for HIT, guidelines from the American Society of Hematology recommend calculation of the 4Ts score to determine the need for laboratory testing. The Choosing Wisely® initiative recommends against laboratory testing in patients with a low probability score of ≤3. In patients with an intermediate or high probability score (≥4), screening with enzyme-linked immunosorbent assay (ELISA) is performed first. If positive, the diagnosis of HIT is confirmed with a functional assay, commonly the serotonin release assay (SRA). Methods: In an effort to increase recognition of HIT, providers at a large academic medical center received a non-interruptive alert in the electronic medical record (EMR) on all patients in whom the platelet count declined by ≥50% starting in Aug 2017. We performed a retrospective evaluation of 1) the number of alerts and 2) all ELISA results obtained with or without an alert, over a 90-day period (Dec 2019 to March 2020). A 4Ts score was calculated by chart review by the first author in real-time as the alert was sent (blinded to ELISA and SRA results). Among those patients with multiple alerts or test orders, the first instance was used for analysis. Demographic and clinical characteristics were reported using frequencies and percentages, means (standard deviation, SD), and medians (interquartile range, IQR). Patients with alerts and ELISA testing ordered were compared with 2 groups: 1) patients with alerts but no ELISA ordered; 2) patients with no alerts but ELISA ordered. Comparisons were performed using chi squared tests, Fisher's exact tests, t-tests and Wilcoxon rank-sum tests as appropriate. Results: In the 90-day observation period, 302 alerts were fired in 270 patients (Figure 1). Thirty alerts (28 patients, 10%) were generated for patients admitted for organ donation or post-stem cell transplantation, for whom platelet count decline was expected. Excluding these patients, there were 272 alerts in 242 patients (approximately 3 alerts per day in a 1,157-bed hospital). Of patients with alerts, 22 (8%) had a platelet count inaccuracy (i.e. platelets clump or another reason) and 18 (7%) did not receive heparin prior to platelet decline, for a cumulative total of 40 (15%) inappropriate alerts. In patients with an alert, the ELISA was ordered more frequently for those with a lower platelet nadir (77x10 9/L vs. 115x10 9/L, p<0.0001) or in those with a thrombotic event (11 patients (17%) vs. 6 patients (4%), p=0.0021) (Table 1). Those without an ELISA ordered were more likely to have a low 4Ts score (23 patients (36%) vs. 81 patients (58%), p<0.0001). In addition to 71 patients with an alert, an ELISA was also ordered for 67 patients without an alert (n=138) (Figure 1). Close to half of ELISA-tested patients had a low 4Ts score (n=51, 46%) (Figure 2). In patients with an alert and ELISA not ordered, 18 (27%) had an intermediate or high 4Ts score. Seven patients were diagnosed with HIT based on a positive SRA, 6 with an alert and 1 without. The alert demonstrated a sensitivity of 86% (95% CI, 59.8-100%) and specificity of 50% (95% CI, 41.8-58.9%) with a positive predictive value of 0.0845 (95% CI, 0.0198-0.1492) and negative predictive value of 0.9851 (95% CI, 0.9560-1.0000). Conclusion: An EMR alert based on platelet count decline had multiple shortcomings including frequent inappropriate firings and a lack of guidance on appropriate indications for testing. This evaluation of institutional testing practices indicates frequent use and misinterpretation of ELISA discordant with evidence-based guidelines. Although prompt diagnosis of HIT is important, alternative strategies for identification of at-risk patients and communication of recommended actions to providers should be considered. Because the 4Ts score includes variables difficult to automate in the EMR, our institution is exploring electronic consultation and real-time expert provider access to overcome these limitations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

855 Development of a novel anti-multiple myeloma chimeric antigen receptor T cell therapy

BackgroundMultiple myeloma is a cancer of plasma cells, wherein the plasma cells begin outgrowing and even suppressing the growth of normal hematopoietic-lineage cells in the blood marrow. It is estimated that nearly 35,000 new cases of multiple myeloma will be diagnosed each year, and over 12,000 individuals will die from multiple myeloma in 2021. The current overall 5-year survival rate of 54% stresses the need for alternative treatment strategies. A chimeric antigen receptor T cell (CAR-T) therapy that targets multiple myeloma surface proteins may have the potential to improve this survival rate.MethodsA commonly associated multiple myeloma antigen was selected, and the extracellular domain of the protein was expressed in an Expi293 cell culture system. The multiple myeloma protein was expressed with and purified by a Hisx6 tag. The purified protein was used to pan a specific scFv phage display library that was generated uniquely for our lab from multiple blood donors. Several clones were identified and sequenced. One clone was selected and verified to be capable of binding the multiple myeloma antigen via ELISA. This anti-multiple myeloma scFv was then subcloned into CAR-T plasmids. This is a second-generation CAR-T using a 4-1BB co-stimulator domain and a CD3ζ intracellular signaling domain was used for this therapy. Further, a cell-fate control gene, LNGFRΔTmpk, was added to the plasmid to combat potential graft versus host disease. CAR-T and lentivirus (LV) plasmids were transfected into HEK cells to produce anti-multiple myeloma CAR-T LV.ResultsThe anti-multiple myeloma CAR LV was transduced into HEK cells and titered via qPCR, which showed the LV prep produced 3.1E8 IU/ml. qPCR also showed there to be 0.407 vector copies per cell on average when using an MOI of 3. Our CAR-T LV was then used to transduce Jurkat cells at an MOI of 3. Using anti-Fab and anti-protein L antibodies, the transduced cells were shown to be expressing the anti- multiple myeloma CAR-T by flow cytometry. After confirming successful transduction and expression, transduced Jurkats (and controls) were co-cultured with multiple myeloma cell lines and incubated for 24 hours. ELISA testing for IFN-γ was performed on the resulting supernatants; specific engagement was demonstrated.ConclusionsThese results show that our anti-multiple myeloma CAR is successfully expressed and is functional. Our future goal is to successfully transduce our anti-multiple myeloma CAR LV into primary T cells and test for functionality in these cells before transitioning into in vivo models.

Non-Invasive Antibody Assessment in Saliva to Determine SARS-CoV-2 Exposure in Young Children

Saliva is a body fluid with hitherto unused potential for the assessment of SARS-CoV-2 antibodies. Specific antibodies can indicate a past SARS-CoV-2 infection and allow to estimate the proportion of individuals with a potential protective immunity. First, we carefully characterized plasma samples obtained from adult control groups with and without prior SARS-CoV-2 infection using certified reference ELISAs. Simultaneously collected saliva samples of confirmed convalescent and negative individuals where then used to validate the herein newly developed ELISA for the detection of SARS-CoV-2 IgG antibodies in saliva. The saliva ELISA was applied to assess SARS-CoV-2 exposure in young children (N = 837) in the age between 1 and 10 years in Tübingen, Germany, towards the end of the first pandemic year 2020. Sensitivity and specificity of the new saliva ELISA was 87% and 100%, respectively. With 12% of all Tübingen children sampled via their respective educational institutions, estimates of SARS-CoV-2 antibody prevalence was 1.6%. Interestingly, only 0.4% preschool kids were positive compared to 3.0% of primary school children. Less than 20% of positive children self-reported symptoms within two months prior to saliva sampling that could be associated - but not exclusively - with a SARS-CoV-2 infection. The saliva ELISA is a valid and suitable protocol to enable population-based surveys for SARS-CoV-2 antibodies. Using non-invasive sampling and saliva ELISA testing, we found that prevalence of SARS-CoV-2 antibodies was significantly lower in young children than in primary school children.

Aquaporin-4 Autoantibody Detection by ELISA: A Retrospective Characterization of a Commonly Used Assay

Objective. Aquaporin-4 (AQP4) serum autoantibodies are detected by a variety of methods. The highest sensitivity is achieved with cell-based assays, but the enzyme-linked immunosorbent assay (ELISA) is still commonly utilized by clinicians worldwide. Methods. We performed a retrospective review to identify all patients at the University of Utah who had AQP4 ELISA testing at ARUP Laboratories from 2010 to 2017. We then reviewed their diagnostic evaluation and final diagnosis based on the ELISA titer result. Results. A total of 750 tests for the AQP4 ELISA were analyzed, and 47 unique patients with positive titers were identified. Less than half of these patients (49%) met the clinical criteria for neuromyelitis optica spectrum disorder (NMOSD). In cases of low positive titers (3.0–7.9 U/mL, n = 19 ), the most common final diagnosis was multiple sclerosis (52.6%). In the moderate positive cohort (8.0–79.9 U/mL, n = 14 ), only a little more than half the cohort (64.3%) had NMOSD. In cases with high positives (80–160 U/mL, n = 14 ), 100% of patients met clinical criteria for NMOSD. Conclusions. Our data illustrates diagnostic uncertainty associated with the AQP4 ELISA, an assay that is still commonly ordered by clinicians despite the availability of more sensitive and specific tests to detect AQP4 autoantibodies in patients suspected of having NMOSD. In particular, low positive titer AQP4 ELISA results are particularly nonspecific for the diagnosis of NMOSD. The importance of accessibility to both sensitive and specific AQP4 testing cannot be overemphasized in clinical practice.

Scrub Typhus – A Threatening Scenario in North Bengal

BACKGROUND Scrub typhus is a mite borne zoonotic bacterial disease caused by Orientia tsutsugamushi. It is transmitted by bite of chiggers of trombiculid mite. Clinical features generally include fever, headache, and myalgia, with or without eschar/rash. People with severe illness may develop organ failure and bleeding which can be fatal if left untreated. This study was done to detect outbreak of cases of scrub typhus in Eastern India. These mites generally live in paddy fields of forested area and people visiting those areas are generally affected. Now a days these mites migrate to urban area resulting in increased incidence of scrub typhus infection in urban area. This study was conducted in collaboration with another institute. The purpose of this study was to find out the incidence of scrub typhus in our area and the relationship between occurrence of scrub typhus and seasonal, age and sex variation. METHODS This study was carried out in our tertiary care hospital with 441 samples for a period of one year (01.01.2019 to 31.12.19). All the blood samples collected from febrile patients were subjected to Weil Felix test. If the titre is > 1 : 160; this was further confirmed by specific IgM testing. Both Weil-Felix tests and IgM scrub typhus positive tests were noted. RESULTS Out of 441 samples, 98 (22.2 %, n = 441) samples were positive for both WeilFelix and scrub typhus IgM by enzyme linked immunosorbent assay (ELISA) testing. Most of the cases were seen in males. Seasonal distribution showed higher cases in the months of September and October. CONCLUSIONS In our study, the highest numbers of scrub typhus cases were found in rural areas, during the harvesting period of July–September specially in monsoon or post monsoon period when there is abundance of mite larva. This infection is also reported high in cases among children in the age group of 1 - 14 years. Patients who tested positive for scrub typhus improved radically with doxycycline. KEYWORDS Orientia Tsutsugamushi, Scrub Typhus, IgM ELISA

Comparing ELISA and LC-MS/MS: A Simple, Targeted Postmortem Blood Screen

A comprehensive screening method that is specific, accurate, and customizable is necessary in any forensic toxicology laboratory. Most laboratories utilize some form of immunoassay testing as it is reliable and sensitive with minimal sample preparation and is relatively inexpensive to simultaneously screen for multiple classes of drugs with different chemical properties. However, accessibility to more specific technology and instrumentation such as mass spectrometry has increased and therefore using immunoassay as the screening method of choice may be revisited. A screening method for 42 drugs in postmortem blood was developed and validated following the Organization of Scientific Area Committees for Forensic Science (OSAC) guidelines for toxicology method validation. The method was developed using minimal sample preparation of postmortem blood consisting only of a protein precipitation. Only two internal standards were used which greatly reduces the cost of implementing this method. Limit of detection (LOD), interference studies, processed sample stability and ion suppression/enhancement were examined. Additionally, over 100 case samples were analyzed by both the current enzyme linked immunosorbent assay (ELISA) testing procedure and the proposed liquid chromatography tandem mass spectrometry (LC-MS/MS) screening method. The comparison determined that the LC/MS-MS method performed as well as or better than the ELISA in nearly all cases. The ability to add additional target drugs increases the laboratory’s scope of analysis as well. This method is ideal for forensic laboratories wishing to improve screening while working within budget constraints.

Laser Scribing Fabrication of Graphitic Carbon Biosensors for Label-Free Detection of Interleukin-6

Interleukin-6 (IL-6) is an important immuno-modulating cytokine playing a pivotal role in inflammatory processes in disease induction and progression. As IL-6 serves as an important indicator of disease state, it is of paramount importance to develop low cost, fast and sensitive improved methods of detection. Here we present an electrochemical immunosensor platform based on the use of highly porous graphitic carbon electrodes fabricated by direct laser writing of commercial polyimide tapes and chemically modified with capture IL-6 antibodies. The unique porous and 3D morphology, as well as the high density of edge planes of the graphitic carbon electrodes, resulted in a fast heterogeneous electron transfer (HET) rate, k0 = 0.13 cm/s. The resulting immunosensor showed a linear response to log of concentration in the working range of 10 to 500 pg/mL, and low limit of detection (LOD) of 5.1 pg/mL IL-6 in phosphate buffer saline. The total test time was approximately 90 min, faster than the time required for ELISA testing. Moreover, the assay did not require additional sample pre-concentration or labelling steps. The immunosensor shelf-life was long, with stable results obtained after 6 weeks of storage at 4 °C, and the selectivity was high, as no response was obtained in the presence of another inflammatory cytokine, Interlukin-4. These results show that laser-fabricated graphitic carbon electrodes can be used as selective and sensitive electrochemical immunosensors and offer a viable option for rapid and low-cost biomarker detection for point-of-care analysis.