Multiplex assessment of SARS-CoV-2 antibodies improves assay sensitivity and correlation with neutralizing antibodies.

OBJECTIVES: Detection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection. METHODS: A cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61). RESULTS: Specificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79-95% agreement with the presence of neutralizing antibodies. CONCLUSIONS: The BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations.

Differential Antibody Response to mRNA COVID-19 Vaccines in Healthy Subjects.

Knowledge about development and duration of virus-specific antibodies after COVID-19 vaccination is important for understanding how to limit the pandemic via vaccination in different populations and societies. However, the clinical utility of postvaccination testing of antibody response and selection of targeted SARS-CoV-2 antigen(s) has not been established. The results of such testing from clinical teams independent from vaccine manufacturers are also limited. Here, we report the initial results of an ongoing clinical study on evaluation of antibody response to four different SARS-CoV-2 antigens after first and second dose of Pfizer and Moderna mRNA vaccines and at later time points. We revealed a peak of antibody induction after the vaccine boosting dose with a gradual decline of antibody levels at later time. Anti-nucleocapsid antibody was not induced by spike protein-encoding vaccines and this may continue to serve as a marker of previous SARS-CoV-2 infection. No differences between the two vaccines in terms of antibody response were revealed. Age and gender dependencies were determined to be minimal within the healthy adult (but not aged) population. Our results suggest that postvaccination testing of antibody response is an important and feasible tool for following people after vaccination and selecting individuals who might require a third dose of vaccine at an earlier time point or persons who may not need a second dose due to previous SARS-CoV-2 infection. IMPORTANCE Now that authorized vaccines for COVID-19 have been widely used, it is important to understand how they induce antivirus antibodies, which antigens are targeted, how long antibodies circulate, and how personal health conditions and age may affect this humoral immunity. Here, we report induction and time course of multiple anti-SARS-CoV-2 antibody responses in healthy individuals immunized with Pfizer and Moderna mRNA vaccines. We also determined the age and gender dependence of the antibody response and compared antibody levels to responses seen in those who have recovered from COVID-19. Our results suggest the importance of screening for antibody response to multiple antigens after vaccination in order to reveal individuals who require early and late additional boosting and those who may not need second dose due to prior SARS-CoV-2 infection.

Low Prevalence of Anterior and Posterior Cruciate Ligament Injuries in Patients With Achondroplasia.

BACKGROUND: Anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) injuries and their subsequent reconstructions are common in the general population, but there has been no research regarding ACL or PCL injuries in patients with achondroplasia, the most common skeletal dysplasia. Our goals were to (1) evaluate the prevalence of ACL and PCL injuries in adolescents and adults with achondroplasia, (2) compare this prevalence with that reported for the general population, (3) determine how many patients with ACL or PCL injuries underwent ligament reconstruction as treatment, and (4) determine patient activity levels as they relate to the rate of ACL/PCL injuries and reconstructions. METHODS: We reviewed medical records of 430 patients with achondroplasia seen in the senior author's clinic from 2002 through 2014. Demographic data were reviewed, as well as any documentation of ACL or PCL injury or reconstruction. We called all 430 patients by telephone, and 148 agreed to participate in our survey, whereas 1 declined. We asked these patients about their history of ACL or PCL injury or reconstruction, as well as current and past physical activity levels. RESULTS: No ACL or PCL injuries were found on chart review. One patient reached by telephone reported an ACL injury that did not require reconstruction. This yielded a theoretical prevalence of 3/430 (0.7%). Of the 148 patients surveyed, 43 (29%) reported low physical activity, 75 (51%) reported moderate physical activity, and 26 (17%) reported high physical activity. There was no significant difference in the rate of ACL injury when stratified by physical activity level (P=0.102). CONCLUSIONS: ACL and PCL injuries and reconstructions are extremely rare in patients with achondroplasia, which cannot be completely ascribed to a low level of physical activity. One possible explanation is that patients with achondroplasia, on an average, have a more anterior tibial slope compared with those without achondroplasia, which decreases the force generated within the ACL and may protect against ACL injury. Further research is needed to explore possible causes. LEVEL OF EVIDENCE: Level IV-retrospective review.

Prevalence of Scoliosis and Thoracolumbar Kyphosis in Patients With Achondroplasia.

STUDY DESIGN: Retrospective chart review, case series. OBJECTIVES: To determine the prevalence of scoliosis and kyphosis in patients with achondroplasia. SUMMARY OF BACKGROUND DATA: There is little published research on the prevalence of scoliosis and thoracolumbar kyphosis in patients with achondroplasia. METHODS: The authors retrospectively reviewed charts of 459 patients with achondroplasia who were seen by the senior author, an orthopedic surgeon, from 1999 through 2013, at a tertiary referral center. After excluding patients who presented after spinal surgery and those who were referred for specific non-spinal issues, 326 patients were included (71%). Cobb angles were measured on lateral and posteroanterior radiographs. Scoliosis was defined as curvature on posteroanterior radiographs greater than 10°; thoracolumbar kyphosis was defined as any kyphotic curvature with an apex between T11 and L2. These data were then stratified by sex, age group (0-2, 3-12, 13-19, 20-40, and >40 years), and severity: within normal limits (≤10°), mild (>10°-25°), moderate (26°-50°), and severe (>50°). RESULTS: The study population consisted of 176 males and 150 females with a mean age of 18 years. Scoliosis was observed in 60%. Thoracolumbar kyphosis was observed in 79%, with 52% exhibiting moderate to severe curvature. CONCLUSIONS: In these patients, the rates of scoliosis and kyphosis were 60% and 79%, respectively, which are much higher than the rates reported in the literature for the general population of children. LEVEL OF EVIDENCE: Level 3 or 4.

The role of clinically significant venous thromboembolism and thromboprophylaxis in pediatric patients with pelvic or femoral fractures.

BACKGROUND: In adults, pelvic and femoral fractures have a known association with venous thromboembolic disease and, thus, thromboprophylaxis is the standard of care. However, similar data for children are scarce, and recommendations for pediatric prophylaxis are less clear. Our goals were to: (1) analyze the predisposing risk factors, prevalence, and outcome (including mortality) of clinically significant venous thromboembolism; (2) investigate the use of thromboprophylaxis in pediatric trauma patients and ages at which it was given; and (3) determine the impact that central venous catheters had on the occurrence of venous thromboembolism. METHODS: We reviewed the records of all pediatric patients with pelvic or femoral fracture admitted to our hospital from 1990 through 2009 for occurrence of venous thromboembolism and related mortality, use and effect of central venous catheters, use of thromboprophylaxis (heparin, warfarin, enoxaparin, or factor-X inhibitors), and patient age at administration. Of the 1782 patients, 948 had electronically searchable medication (and device) records. Ninety-five percent confidence intervals were found for all proportions with sample sizes >100, and an unpaired t test was used to compare the average age at which thromboprophylaxis was given with the average age of the total population. RESULTS: Of the 1782 patients, there were 3 (0.17%) diagnoses of deep vein thrombosis and no diagnoses of pulmonary embolism; there was no related mortality. Of the medication subset (948 patients) only 83 (8.8%) received some type of thromboprophylaxis. The average age of patients given thromboprophylaxis was 14.65 years (SD, 2.34). No central venous catheter was associated with any of the patients who had a venous thromboembolic event. CONCLUSIONS: Thromboprophylaxis was used only occasionally at our institution; >91% of patients did not receive such treatment. No morbidity or mortality was reported related to venous thromboembolism in pediatric patients with femur or pelvic fracture for whom thromboprophylaxis was used. LEVEL OF EVIDENCE: Level II, retrospective study.

Laminectomy in patients with achondroplasia: the impact of time to surgery on long-term function.

STUDY DESIGN: Retrospective analysis, survey. OBJECTIVE: To describe a cohort of individuals with achondroplasia undergoing thoracolumbar laminectomy and to examine if shorter time to surgery was related to improvement in long-term functional outcome. SUMMARY OF BACKGROUND DATA: Data on the long-term benefits of laminectomy are mixed for such patients. Earlier intervention may be associated with greater likelihood of long-term benefit, but quantified data are lacking. METHODS: We retrospectively studied 49 patients with achondroplasia who underwent primary laminectomy for spinal stenosis. Patients completed a questionnaire to assess symptoms, walking distance, and independence (per Modified Rankin Scale), before surgery and currently. Responses were analyzed for the likelihood of improved walking distance or Rankin level. RESULTS: Our patients had the following mean values: age, 37.7 ± 10.6 years; body mass index, 31.8 ± 5.5; symptom duration, 74.0 ± 100.1 months; preoperative symptom severity score, 2.7 ± 1.0 points; mean changes in blocks walked, +0.39 ± 2.0; and Rankin level, +0.08 ± 1.47. Patients with a time-to-surgery interval of <6 months were 7.13 times (95% confidence interval [CI], 1.39-36.66) more likely to experience improvement in walking distance and 4.00 times (95% CI, 1.05-15.21) more likely to experience Rankin level improvement than patients whose interval was >6 months. Intervals of up to 12 and 24 months were associated with increased likelihoods of 4.95 (95% CI, 1.41-17.41) and 3.43 (95% CI, 1.05-11.22), respectively, of improved walking distance compared with those with longer time-to surgery intervals, but those Rankin level improvements were not statistically significant. CONCLUSION: Time from symptom onset to surgery in patients with achondroplasia is an important predictor of long-term functional outcome. For sustained long-term postsurgical improvement, the window of opportunity might be relatively narrow. Patients with achondroplasia should seek medical advice for spinal stenotic symptoms as soon as possible.

Individualizing combination of two antiproliferative immunosuppressants with pharmacodynamic modeling of stimulated lymphocyte responses.

BACKGROUND: Unpredictable serious adverse events (SAE) of immunosuppression, e.g. nephrotoxicity, with the nephrotoxic immunosuppressants have fostered interest in alternative regimens, which contain two antiproliferative agents, and individualized therapy. However, titration of such combinations to individual needs is not understood. SPECIFIC AIM: To determine concentration (C) mixtures of mycophenolate mofetil (MMF) and sirolimus (SRL), which produce half-maximal inhibitory effect (EC(50)) on human lymphocytes from individual subjects. METHODS: Concentration mixtures of MMF (0-5 mug/ml) and SRL (0-30 ng/ml) were incubated with whole blood from each of five healthy human subjects. The intracellular cytokines IL-2, TNF-alpha, and IFN-gamma were measured in PMA-ionomycin-stimulated T-cells (CD4+), while CD54, CD95, CD86, CD25, CD69, and CD71 were measured in pokeweed mitogen-stimulated B-cells, by flow cytometry. Pharmacodynamic (PD) relationships were evaluated using Hill equations modified for single and multidrug regimens, and expressed as EC(50) for each target receptor. RESULTS: No change was seen in the expression of the T-cell cytokines with either MMF or SRL. Each B-cell receptor was inhibited with increasing concentrations of either MMF or SRL. Each B-cell receptor was also inhibited half-maximally at lower concentrations of MMF in the presence of SRL, than with either agent alone, for the test population of five subjects together, and for each of five individual subjects. However, each subject showed distinctly different amounts of MMF and SRL that needed to be present together, in order to produce an identical inhibitory effect on lymphocyte function. CONCLUSIONS: PD analysis of biological effect can potentially predict optimal concentration mixtures of two immunosuppressants for individual recipients, and enhance rejection prophylaxis and safety. While this holds promise for drug development efforts, clinical application must await correlation of lymphocyte markers with post-transplant clinical outcomes.