The COVID‐19 pandemic poses an immense need for accurate, sensitive and high‐throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high‐throughput multiplex bead‐based serological assay.

Background: Declining humoral immunity in COVID-19 patients and possibility of reinfections has raised concern. Mucosal immunity particularly salivary antibodies could be short-lived. However, long-term studies are sparse. Methods: Using a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (n=74, Cohort 1), undiagnosed individuals with self-reported questionnaires (n=147, Cohort 2), and individuals sampled pre-pandemic time (n= 35, Cohort 3). Results: Salivary IgG antibody responses in Cohort 1 (mainly mild COVID-19) were detectable up to 9 month recovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in saliva in majority as seen in blood serology. Salivary IgA was rarely detected at this timepoint. In Cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19 like symptoms. Salivary IgG also tolerated temperature and detergent pre-treatments. Conclusions: Unlike SARS-CoV-2 salivary IgA that appeared short-lived, the specific IgG in saliva appears stable even after mild COVID-19 as noted for blood serology. The non-invasive saliva-based SARS-Cov-2 antibody testing with self-collection at homes may thus serve as a complementary alternative to conventional blood serology.

Background: Amniocentesis (AC) is the most commonly used invasive prenatal test. The aim of this study was to determine which were the most common indications for AC, chromosomal abnormalities in relation to the age of the mother and complications as a result of AC. Methods: This is a retrospective thirteen-year cohort study including pregnant women who underwent AC in the period from 2008 to 2020. Patients were divided into two groups: below and above 35 years of age of mother at the time of AC. Results: During study period 2213 AC were performed, out of which 759 (34.29%) were in mother below, and 1454 (65.70%) above 35 years of age (p < 0.00001). The most common indication for AC in group above 35 years of age was advanced maternal age (93.9%), in group below 35 years of age was familial chromosomal diseases (29.91%). In all indications there is a statistically significant difference between the two age groups of pregnant women, all these indications are more prevalent in the group of pregnant women younger than 35 years (p < 0.00001). The most common chromosomal abnormalities in both groups was Down syndrome from numerical aberrations, and from structural inversion of chromosome 9. Short term complications were spontaneous abortion in 26 (1.17%), missed abortion in 4 (0.18%) and premature rupture of membranes in 4 (0.18%) cases. Conclusion: Advanced maternal age, demonstrated the strongest association with the uptake of AC, trisomy 21 was the most prevalent abnormal finding. Complications of AC are not significant compared to the general population of pregnant women.

Public health strategies to contain the pandemic continue to vary markedly across the world. In Sweden, compared to most advanced economies, social restrictions have primarily relied upon voluntary adherence to a set of recommendations and strict lockdowns have not been enforced. To better understand the development of humoral immunity to SARS-CoV-2 in the Stockholm population before the start of mass vaccinations, healthy blood donors and pregnant women (n=4,100) were sampled at random between 14th March-11th December 2020. All individuals (n=200/sampling week) were screened for anti-SARS-CoV-2 spike (S) trimer- and RBD-specific IgG responses with highly sensitive and specific ELISA assays, and the results were compared with those from historical controls (n=595). Data were modelled using a probabilistic Bayesian framework that considered individual responses to both antigens. We found that after a steep rise at the start of the pandemic, the seroprevalence trajectory increased steadily in approach to the winter second-wave of infections, approaching 15% of all individuals surveyed by 11th December. In agreement with the high transmission rate observed in the Stockholm area, seroprevalence in this cohort of active adults increased during the 9 months from the start of the outbreak, but was far from that required for herd immunity at the end of 2020.

Abstract Serology is critical for understanding pathogen-specific immune responses, but is fraught with difficulty, not least because the strength of antibody (Ab) response varies greatly between individuals and mild infections generally generate lower Ab titers1–3. We used robust IgM, IgG and IgA Ab tests to evaluate anti-SARS-CoV-2 responses in individuals PCR+ for virus RNA (n=105) representing different categories of disease severity, including mild cases. All PCR+ individuals in the study became IgG-positive against pre-fusion trimers of the virus spike (S) glycoprotein, but titers varied greatly. Elevated IgA, IL-6 and neutralizing responses were present in intensive care patients. Additionally, blood donors and pregnant women (n=2,900) sampled throughout the first wave of the pandemic in Stockholm, Sweden, further demonstrated that anti-S IgG titers differed several orders of magnitude between individuals, with an increase of low titer values present in the population at later time points4,5. To improve upon current methods to identify low titers and extend the utility of individual measures6,7, we used our PCR+ individual data to train machine learning algorithms to assign likelihood of past infection. Using these tools that assigned probability to individual responses against S and the receptor binding domain (RBD), we report SARS-CoV-2-specific IgG in 13.7% of healthy donors five months after the peak of spring COVID-19 deaths, when mortality and ICU occupancy in the country due to the virus were at low levels. These data further our understanding of antibody responses to the virus and provide solutions to problems in serology data analysis. Significance statement Antibody testing provides critical clinical and epidemiological information during an emerging disease pandemic. We developed robust SARS-CoV-2 IgM, IgG and IgA antibody tests and profiled COVID-19 patients and exposed individuals throughout the outbreak in Stockholm, Sweden, where full societal lockdown was not employed. As well as elucidating several disease immunophenotypes, our data highlight the challenge of identifying low IgG titer individuals, who comprise a significant proportion of the population following mild/asymptomatic infection, especially as antibody titers wane following peak responses. To provide a solution to this, we used SARS-CoV-2 PCR+ individual data to develop machine learning approaches that assigned likelihood of past infection to blood donors and pregnant women, improving the accuracy and utility of individual and population-level Ab measures.

Background: The aim of this study is to compare the antenatal care, body weight, and weight gain in pregnancy between the adolescent and adult pregnancies and, thus, examine the impact of adolescence on the studied parameters. Methods: This prospective study includes 300 pregnant women who were the patients of University Clinical Center Tuzla, Clinic for Gynecology and Obstetrics from January 2011 to December 2014. The women were divided into two groups: an experimental group consisted of 150 adolescent pregnant women aged 13–19 years and a control group consisted of 150 adult pregnant women aged 20–35 years. The following parameters were analyzed: age of pregnant women, number of antenatal controls in pregnancy, prepregnancy body weight, weight gain in pregnancy, parity, and obstetric history data. Results: A significantly higher number of adolescent pregnant women belongs to a subgroup from one to two examinations during pregnancy (P < 0.000013) and to subgroups from three to five examinations (P < 0.000001). A significantly smaller number of adolescent pregnant women performed their first antenatal control in the first 2 lunar months (P < 0.01). A subgroup with optimal body weight (from 51 to 69 kg) are the most prevalent among adolescent pregnant women (P < 0.000001). A significantly larger number of adolescent pregnant women had an optimal weight gain of 7.8 to 12.99 kg (P < 0.001). Conclusions: The adolescent pregnant women have suboptimal antenatal care, which could lead to adverse maternal and birth outcomes, but have optimal body weight and weight gain during pregnancy.

Aim To investigate clinical and obstetrical characteristics, an outcome and a prognosis for pregnant women with diagnosed and treated genital or extragenital cancer and their newborns. Methods This retrospective cohort study included pregnant and childbearing women with a history of cancer diagnosed before pregnancy during the period between 1 January 2014 and 31 December 2018. Data related to the course of pregnancy and childbirth were collected from medical records (mothers' disease history and partogram). The analysis covered clinical and histopathological characteristics of cancers, type of the treatment (surgery, chemotherapy, radiotherapy), demographic data, obstetric characteristics, comorbidities of women, and outcome of the newborns. Results The study recorded 18 414 deliveries, of which 30 (0.16%) were pregnancies in women who had been diagnosed and treated earlier for genital or extragenital cancer. The average age of the women at the time of delivery was 29.43±5.97 years. There were six (20%) women with genital and 24 (80%) with extragenital cancer. The most frequent extra genital cancer was Hodgkin lymphoma, in eight (26.6%) cases; ovarian cancer was the most frequent genital cancer, in four (13.3%) cases. The average time span from the cancer diagnosis and start of the treatment to the delivery was 59.2±44.4 months (5 years) (range 12 months - 15 years). Two (6.6%) women died. Conclusion Our data demonstrate a favourable obstetric and neonatal outcome for women who have survived cancer.

Antibody responses vary widely between individuals1, complicating the correct classification of low-titer measurements using conventional assay cut-offs. We found all participants in a clinically diverse cohort of SARS-CoV-2 PCR+ individuals (n=105) – and n=33 PCR+ hospital staff – to have detectable IgG specific for pre-fusion-stabilized spike (S) glycoprotein trimers, while 98% of persons had IgG specific for the receptor-binding domain (RBD). However, anti-viral IgG levels differed by several orders of magnitude between individuals and were associated with disease severity, with critically ill patients displaying the highest anti-viral antibody titers and strongest in vitro neutralizing responses. Parallel analysis of random healthy blood donors and pregnant women (n=1,000) of unknown serostatus, further demonstrated highly variable IgG titers amongst seroconverters, although these were generally lower than in hospitalized patients and included several measurements that scored between the classical 3 and 6SD assay cut-offs. Since the correct classification of seropositivity is critical for individual- and population-level metrics, we compared different probabilistic algorithms for their ability to assign likelihood of past infection. To do this, we used tandem anti-S and -RBD IgG responses from our PCR+ individuals (n=138) and a large cohort of historical negative controls (n=595) as training data, and generated an equal-weighted learner from the output of support vector machines and linear discriminant analysis. Applied to test samples, this approach provided a more quantitative way to interpret anti-viral titers over a large continuum, scrutinizing measurements overlapping the negative control background more closely and offering a probability-based diagnosis with potential clinical utility. Especially as most SARS-CoV-2 infections result in asymptomatic or mild disease, these platform-independent approaches improve individual and epidemiological estimates of seropositivity, critical for effective management of the pandemic and monitoring the response to vaccination.

Serological studies are critical for understanding pathogen-specific immune responses and informing public health measures (1,2). By developing highly sensitive and specific trimeric spike (S)-based antibody tests, we report IgM, IgG and IgA responses to SARS-CoV-2 in COVID-19 patients (n=105) representing different categories of disease severity. All patients surveyed were IgG positive against S. Elevated anti-SARS-CoV-2 antibody levels were associated with hospitalization, with IgA titers, increased circulating IL-6 and strong neutralizing responses indicative of intensive care status. Antibody-positive blood donors and pregnant women sampled during the pandemic in Stockholm, Sweden (weeks 14-25), displayed on average lower titers and weaker neutralizing responses compared to patients; however, inter-individual anti-viral IgG titers differed up to 1,000-fold. To provide more accurate estimates of seroprevalence, given the frequency of weak responders and the limitations associated with the dichotomization of a continuous variable (3,4), we used a Bayesian approach to assign likelihood of past infection without setting an assay cut-off. Analysis of blood donors (n=1,000) and pregnant women (n=900) sampled weekly demonstrated SARS-CoV-2-specific IgG in 7.2% (95% Bayesian CI [5.1-9.5]) of individuals two months after the peak of spring 2020 COVID-19 deaths. Seroprevalence in these otherwise healthy cohorts increased steeply before beginning to level-off, following the same trajectory as the Stockholm region deaths over this time period.

Background: To investigate the total survival of low birth weight infants (LBWIs) in the Federation of Bosnia and Herzegovina (FB and H) and selected by subgroups of birth weight (BW) and gestational age (GA). Methods: This cross-sectional study included newborns of both genders, GA of 22–42 weeks and BW of less than 2500 g of 10 cantons territory of the FB and H. In the examined period, 22,897 children were born in the FB and H, of which 669 (2.9%) had BW less than 2500 g. Results: Surviving of LBWIs in the FB and H out of the 669 LBWIs in the first level perinatal healthcare institutions (PHI) was 29 (4.3%), the second level was 286 (42.8%), and the third level was 354 (52.9%). The total stillborn rate was 3.9%. The overall perinatal mortality rate for all levels of PHI was 8.6%. The overall rate of early neonatal mortality of LBWIs in all three levels of PHI in the FB and H was 12.7%. By the end of the first month of life (up to 28 days) and to the end of the neonatal period, 385 (57.5%) of LBWIs survived, and 284 (42.4%) died. The LBWIs by subgroups of BW up to 28 days had lower survival rates in second-level PHI than infants of the same BW subgroups (500–999 and 1000–1499) treated in third-level PHI (P = 0.0089 and P = 0.004). Conclusions: Our results show that B and H belongs to developing countries according to perinatal mortality. A unique database system is necessary to follow progress and trends.

Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with an increased risk of developing cardiovascular diseases as a result of complex interaction between traditional risk factors, chronic inflammation and specific impact of antibodies on endothelium. There are very limited data regarding level of physical activity (PA) in APS patients.To analyze different domains of PA in Serbian APS patients and their possible relationship to clinical and laboratory criteria of the main disease.From a large Serbian APS database comprehending 527 APS patients (371 Primary – PAPS, and 156 APS associated with other autoimmune diseases, predominantly systemic lupus erythematosus (SLE)) we interviewed 51 APS patients, age range of 15-69 years: 29 patients with primary APS (PAPS), 25 women, 4 men, age 44±11.50, and 22 APS/SLE, 18 women, 4 men, age 48.41±11.75, using a long form of The International Physical Activity Questionnaire (IPAQ), translated onto Serbian language. Data on last seven days of PA divided onto leisure time PA, domestic and gardening (yard) activities, work-related PA and transport-related PA were acquired, and proposed scoring method was used. Based on the level of PA, patients were categorized to low, moderate or high level of PA. For the purpose of insight into atherosclerotic progression, we performed color Doppler scan of carotid arteries in all patients and presence of atherosclerotic plaques has been notified.Average total PA score was 7706.18±11771.97 MET-minutes/week. The greatest average values for different PA domains were for work (2733.21±6158.66 MET-minutes/week) and domestic/garden/yard (2522.31±3847.24 MET-minutes/week) and the lowest scores achieved in leisure time (500.87±695.45 MET-minutes/week). Majority of Serbian APS patients had low or moderate level of PA (37.3%, 43.1%, respectively) whereas lowest percentage was in high category of PA (19.6%). All domains of PA were significantly negatively correlated to age and BMI. There were no significant difference regarding PA scores between PAPS and APS/SLE patients. Although higher percentage of PAPS patients had high level of PA (27.65 compared to 9.1% of SLE/APS), the overall difference was not significant. There was no significant difference regarding antiphospholipid antibody (aPL) type or thrombotic/obstetric events presence. Significant difference occurred regarding presence of carotid arteries plaques. APS patients with lower PA scores had significantly higher prevalence of carotid arteries plaque especially for PA in transportation (p=0.004), and total PA (p=0.025)Serbian APS patients at younger age, tend to have low or moderate level of PA, with the lowest level of activity in leisure time. Low level of PA was undoubtedly related to progression of atherosclerosis in these patients, emphasizing a need for PA promotion in APS.Aleksandra Djokovic Speakers bureau: KRKA, Astra Zeneca, Actavis, Ljudmila Stojanovich: None declared, Natasa Stanisavljevic: None declared, Gordana Bogdanovic: None declared, Sandra Djokic: None declared

Background: Borrelia burgdorferi and tick-borne encephalitis (TBE) virus are 2 types of tick-borne pathogens that can cause central nervous system infection. Routine diagnostics have so far included analysis of cerebrospinal fluid (CSF) cell numbers, CSF serology for Borrelia burgdorferi and serum serology for TBE virus. However, early diagnosis may be difficult based on antibody detection which takes time to analyze, and with the possibility of false negative results, thus delaying treatment. Cytokine analyses are becoming increasingly available in clinical routine care and may offer important information. Methods: Fifteen cytokines and chemokines were measured in the CSF from the diagnostic lumbar puncture of 37 children with TBE, 34 children with neuroborreliosis and 19 children without evidence of central nervous system infection, using Luminex technology. Results: Significantly higher levels of proinflammatory interleukin-6 were detected in the samples from TBE-infected children, when compared with neuroborreliosis or controls. In comparison, children with neuroborreliosis had significantly higher levels of interleukin-7, interleukin-8, interleukin-10, and interleukin-13 when compared with TBE infected or controls. Furthermore, the ratio between interleukin-6 and interleukin-10 was significantly different between the 2 types of tick-borne infections. Conclusions: The interleukin-6/interleukin-10 ratio can be used as a rapid diagnostic cue upon suspected tick-borne infection, enabling fast and correct treatment. Also, in serology-negative results, such information may strengthen a clinical suspicion.