Qualitative Variation among Commercial Immunoassays for Detection of Measles-Specific IgG.

Measurement of measles virus-specific IgG is used to assess presumptive evidence of immunity among immunocompetent individuals with uncertain immune or vaccination status. False-negative test results may lead to unnecessary quarantine and exclusion from activities such as employment, education, and travel or result in unnecessary revaccination. In contrast, false-positive results may fail to identify susceptible individuals and promote spread of disease by those who are exposed and unprotected. To better understand the performance characteristics of tests to detect measles IgG, we compared five widely used, commercially available measles IgG test platforms using a set of 223 well-characterized serum samples. Measles virus neutralizing antibodies were also measured by in vitro plaque reduction neutralization, the gold standard method, and compared to IgG test results. Discrepant results were observed for samples in the low-positive ranges of the most sensitive tests, but there was good agreement across platforms for IgG-negative sera and for samples with intermediate to high levels of IgG. False-negative test results occurred in approximately 11% of sera, which had low levels of neutralizing antibody.

Determination of Voriconazole Concentrations in Serum by GC-MS.

BACKGROUND: Voriconazole is a broad spectrum triazole antifungal drug used to treat systemic fungal infections. Therapeutic drug monitoring of voriconazole is necessary for achieving maximal efficiency without inducing toxic side effects. Other publications have reported methods for measuring voriconazole in serum using high-performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Here, we report for the first time a method for the measurement of voriconazole in serum samples using gas chromatography mass spectrometry (GC-MS). METHODS: Protein precipitation with methanol was used to extract the antifungal that was derivatized with BSTFA (Sigma-Aldrich, St. Louis, MO) and analyzed by GC-MS. Linearity, sensitivity, precision, accuracy, and drug interferences were evaluated for this assay. RESULTS: Our method was linear up to 10 µg/ml of voriconazole. The LOQ was determined to be 0.4 µg/ml. CV for between-day precision was <12%. Correlation with an established LC-MS/MS yielded a R2 of 0.96. Tested drugs did not result in >10% error in measurement. CONCLUSION: To our knowledge, we report here the first GC-MS method for voriconazole measurement with acceptable performance. We hope that this method allows clinical laboratories without HPLC or LC-MS/MS instrumentation to measure voriconazole.

Preliminary Investigation into the Prevalence of G6PD Deficiency in a Pediatric African American Population Using a Near-Patient Diagnostic Platform.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in the African American population. We identified eighteen G6PD-deficient samples (9%) in a study of residual, de-identified whole blood specimens from 200 African American pediatric patients using a point-of-care instrument. This highlights the possibility of a rapid time to result for G6PD testing, which can be valuable in some clinical scenarios.

Human cytomegalovirus protein pUL117 targets the mini-chromosome maintenance complex and suppresses cellular DNA synthesis.

Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.

Impact of the AACC Global Laboratory Quality Initiative in Partnership with Professional Societies and Universities in Latin America and the Caribbean.

The Global Lab Quality Initiative (GLQI), formerly known as the Emerging Countries program, was funded through a generous endowment from the Wallace H. Coulter Foundation. The aims of GLQI are to develop and implement innovative programs to promote education and training in laboratory medicine for low- or lower middle-income countries worldwide. From its inception in 2010, the GLQI was focused solely on the Latin America and Caribbean (LAC) region under the purview of AACC's Latin American Working Group (LAWG), the members of which have strong ties to the region thereby facilitating the partnerships with national societies. The LAWG has provided in-person workshops in the LAC countries, at the AACC Annual Scientific Meeting, and on-demand webinars. The LAWG aims to implement the GLQI aims in the LAC region. In-person workshops are based on best-practice recommendations and sources such as Clinical Laboratory Standard Institute guidelines and supplemented with professional experiences of the LAWG's lecturers and local experts of the countries visited. In 2015, the GLQI expanded to other regions of the world. Here we report the experience of the LAWG workshops, results of participant surveys, in-person visits to laboratories post-workshop, and the lessons learned throughout the years across different geographic areas. We are hopeful this report provides insights into the challenges and successes of the LAWG in LAC to help support the expansion of the GLQI.

Dataset for longitudinal evaluation of the Abbott ARCHITECT SARS-CoV-2 IgM and IgG assays in a pediatric population divided by age.

BACKGROUND: SARS-CoV-2 infection in children does not seem to follow the same pattern as in adults. Limited information is published on the level of antibody production and the duration of antibody response in children with COVID-19. Moreover, it is unknown if all children have a similar immune response to the infection, or if there are age dependent differences. In these data, we look at the IgM and IgG levels and duration of two age groups infected by the SARS-CoV-2 virus. METHODS: Residual laboratory specimens from pediatric patients positive for SARS-CoV-2 infection were tested for IgM and IgG against SARS-CoV-2 using an automated Abbott ARCHITECT i1000. We tested 181 specimens from 41 patients with a positive molecular result. Data was grouped either as time after nucleic acid amplification test (NAAT) or time after symptom onset. Patient samples were divided into 2 age groups: 0 to 11 years old and 12 to 19 years old. The assays detect IgM against the spike protein and IgG against the nucleocapsid protein.

Longitudinal evaluation of the Abbott ARCHITECT SARS-CoV-2 IgM and IgG assays in a pediatric population.

BACKGROUND: Clinical laboratory testing has been an essential part of COVID-19 management. Serology can provide valuable information regarding a patient's exposure to virus, and may have a larger role to play as vaccines becomes available. Limited data is available on the serological response in pediatric patients. Here we investigate the use of one manufacturer's commercial assays for detecting IgM and IgG in an exclusively pediatric population. METHODS: Abbott SARS-CoV-2 IgM and IgG assays were performed on an Abbott ARCHITECT i1000. For specificity studies, we tested 78 patient specimens collected before the COVID-19 pandemic, and 66 specimens from patients who tested negative for SARS-CoV-2 nucleic acid amplification test (NAAT) during the COVID-19 pandemic. For sensitivity we tested 181 specimens from 41 patients with a positive NAAT result. Precision data was acquired for 20 days. RESULTS: For IgM, the highest qualitative positive agreement with molecular results was observed to be 15-30 days after a positive NAAT result or after symptom onset. For IgG, the highest positive agreement was 31-60 days after a positive NAAT result or 61-90 days after the start of symptoms. IgM started to decline 30 days after NAAT results and faded by 90 days. IgG started to decrease 60 days after a positive NAAT result. CONCLUSION: The Abbott IgM and IgG assays have negative agreements of 98.7-100% relative to NAAT results. The IgM and IgG levels assayed by these methods start to decline months after positive molecular results and onset of symptoms in a pediatric population.

Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit.

In higher eukaryotic organisms, the checkpoint kinase 1 (Chk1) contributes essential functions to both cell cycle and checkpoint control. Chk1 executes these functions, in part, by targeting the Cdc25A protein phosphatase for ubiquitin-mediated proteolysis. In response to genotoxic stress, Chk1 is phosphorylated on serines 317 (S317) and 345 (S345) by the ataxia-telangiectasia-related (ATR) protein kinase. Phosphorylation of Chk1 on these C-terminal serine residues is used as an indicator of Chk1 activation in vivo. Here, we report that inhibition of Chk1 kinase activity paradoxically leads to the accumulation of S317- and S345-phosphorylated Chk1 in vivo and that ATR catalyzes Chk1 phosphorylation under these conditions. We demonstrate that Chk1 phosphorylation by ATR is antagonized by protein phosphatase 2A (PP2A). Importantly, dephosphorylation of Chk1 by PP2A is regulated, in part, by the kinase activity of Chk1. We propose that the ATR-Chk1-PP2A regulatory circuit functions to keep Chk1 in a low-activity state during an unperturbed cell division cycle but at the same time keeps Chk1 primed to respond rapidly in the event that cells encounter genotoxic stress.

Probabilistic Modeling Approach to Reducing Healthcare Costs With Reflex Testing.

OBJECTIVE: Statistical methods can be utilized to optimize the order for reflex diagnostic testing to attenuate patient and hospital costs without affecting quality of care. Our objective is to demonstrate the method of developing an order for testing and to apply this method to an illustrative example. METHODS: An algorithm was developed for minimizing costs for any given number of diagnostic tests, and it was retrospectively applied to a sample. RESULTS: The actual scenario of using both tests on all patients was compared to 2 other hypothetical reflex testing approaches: all patients are given 1 test, and those patients who tested negative were then given the second test. The 2 scenarios would have saved 37.1% and 17.4% in testing costs, respectively. CONCLUSION: These calculations could be applied to numerous situations to reduce costs for patients and hospitals. We propose that this methodology would be best used in conjunction with any existing quality improvement initiatives.

Induction of p21 and p27 expression by amino acid deprivation of HepG2 human hepatoma cells involves mRNA stabilization.

mRNA abundance for a number of genes is increased by amino acid limitation. From an array screening study in HepG2 human hepatoma cells, it was established that one set of genes affected by amino acid availability is the set associated with cell-cycle control. The present study describes the increased expression of both mRNA and protein for the cyclin-dependent kinase inhibitors p21 and p27 in response to deprivation of HepG2 cells for a single essential amino acid, histidine. The increase in p21 and p27 mRNA content depended on de novo protein synthesis and involved a post-transcriptional mRNA stabilization component. For p21, increase in mRNA by histidine depletion appeared to be independent of p53 transactivation, and the absolute level of p53 protein was unaffected by this treatment. Histidine limitation caused an increase in the phosphorylation of ERK1/ERK2 (extracellular-signal-regulated kinase), and inhibition of the ERK signal transduction pathway resulted in a reduction in the starvation-dependent increase in p21 mRNA. Blockade of the phosphoinositide 3-kinase and mTOR (mammalian target of rapamycin) pathways also blunted the increase in p21 mRNA content. These results document the amino acid-dependent control of the synthesis of specific cell-cycle regulators and help to explain the block at G1 phase after amino acid limitation.

A 14-year old girl with reversible hypoglycemic episodes: the role of ASVS.

We present the case of a 14-year-old female who experienced several episodes of reversible altered mental status triggered by hypoglycemia. Following endocrine investigation, she was diagnosed with insulinoma. Insulinoma, a rare, differentiated, and functioning neuroendocrine tumor of the pancreas overproduces insulin, thus leading to hypoglycemic episodes. Conventional imaging failed to detect the lesion; therefore, arterial calcium stimulation with venous sampling (ASVS) was used for preoperative localization. The patient recovered without complications after surgical enucleation of the tumor. The ASVS is a useful method for localizing insulinomas when conventional imaging techniques fail, and can help reduce morbidities associated with surgical excision.

Universal cholesterol screening of children in community-based ambulatory pediatric clinics.

BACKGROUND: Early identification and treatment of individuals with elevated levels of atherogenic cholesterol have been shown to be effective and safe in reducing morbidity and mortality, especially in familial hypercholesterolemia. To better inform providers and identify children and adolescents at risk of premature cardiovascular disease, in November 2011, the National Heart, Lung, and Blood Institute (NHLBI) published guidelines recommending cholesterol screening of all children aged between 9 to 11 and 17 to 21 years regardless of the child's general health or the presence or the absence of cardiovascular disease risk factors. OBJECTIVE: To compare the number of 9- to 11-year-old children screened for hypercholesterolemia in 5 community-based ambulatory pediatric clinics before and after publication of the NHLBI's guidelines. METHODS: Practice demographics, screening frequency, and test results for each clinic were collected before and after publication of the NHLBI's recommendation. Provider education was provided between measures. RESULTS: Of all eligible 9- to 11-year-old children, 489 (17.1%) were screened before and 686 (20.1%) after the NHLBI's guidelines and provider education. CONCLUSIONS: Baseline rates of lipid screening for the 5 community-based ambulatory pediatric clinics were higher than those previously reported and increased significantly after publication of the NHLBI's recommendations and provider education. However, overall screening rates remained low. Given the high prevalence of premature cardiovascular disease associated with atherogenic cholesterol, especially familial hypercholesterolemia, additional strategies are needed to improve screening rates.

Predicting respiratory distress syndrome using gestational age and lamellar body count.

OBJECTIVES: To design a predictive model for assessing the risk of developing respiratory distress syndrome (RDS) using gestational age (GA) and lamellar body counts (LBC). DESIGN AND METHODS: LBCs and patient outcome data was obtained from five medical centers. A total of 223 patients were included in this study; 19 gave birth to infants that developed RDS, 204 gave birth to infants that were unaffected. The absolute risk and odds ratios of an infant developing RDS as a function of GA and LBC were calculated. Logistic analysis was used to model the odds of RDS as a function of GA and LBC. RESULTS: The odds of RDS decreased for each increasing week of GA and decreased with increase in the LBC. GA-specific LBC cutoffs are provided for sensitivities between 84 and 100%. The bias adjusted area under the ROC curve for the classification of RDS, based on GA and LBC, was 0.906 using the logistic model and 0.746 using a single cutoff of LBC (50,000/µL) to classify immaturity. CONCLUSIONS: GA-specific risk assessment and GA-specific cutoffs provide increased sensitivity and specificity in the evaluation of fetal lung maturity.

Biomarker tests for fetal lung maturity.

The production of surfactant is a key step in fetal lung development. Surfactant decreases alveolar surface tension, thereby preventing alveolar collapse and allowing efficient gas exchange. The lack of adequate amounts of lung surfactant results in respiratory distress syndrome. Tests that assess surfactant concentrations in amniotic fluid are good predictors of infants that will not develop respiratory distress syndrome. The most frequently used test to assess fetal lung maturity (TDx FLM II) will not be available after December 2011. Therefore, we review the currently available tests for fetal lung maturity including lecithin:sphingomyelin ratio, phosphatidyl glycerol, surfactant:albumin ratio and lamellar body counts. Herein, we discuss their clinical utility and consider a suitable replacement for the future.