Paired Serum Specimens Research Articles

B-079 Characterization of an isoelectric focusing method for detection of IgG oligoclonal banding in paired cerebrospinal fluid and serum specimens

Abstract Background Qualitative assessment of IgG-specific oligoclonal banding (OCB) in paired serum and cerebrospinal fluid (CSF) samples is useful for evaluating inflammatory conditions of the central nervous system such as multiple sclerosis. Here we characterized the performance of an IgG-specific method using isoelectric focusing on agarose gel matrix for the detection of OCB in paired serum and CSF samples. Methods Specimens of 20 paired, simultaneously collected CSF and serum patient samples were tested on Sebia (Hydrasys 2 System) provided by the National Institutes of Health (NIH) Diagnostic Laboratories. We tested these paired samples using the Helena IgG IFE-20 kit (SPIFE® Touch) method and the results were read by two independent individuals blinded to each other’s assessments. The results were compared to the NIH results for agreement. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were calculated. Analytical sensitivity of the Helena assay was assessed via serial dilutions of a CSF OCB positive sample (total IgG 4.5 mg/dL). Results Figure 1 shows serum and CSF pairs for the control (A), two example positive OCB patients (B & D) and a negative OCB patient (C). The Helena assay was concordant with Sebia for 11/12 positive OCB patients (PPA 91.6%) and 8/8 negative subjects (NPA 100%). 20-fold was the highest dilution that remained positive. Conclusions OCB detection using paired serum and CSF samples and isoelectric focusing electrophoresis is considered the preferred method. The Helena IgG IFE-20 assay is such a method and showed high PPA and NPA concordance with the Sebia System. Thus, the Helena OCB assay can be considered an accurate and analytically sensitive method for assessing OCB in serum and CSF.

B-311 Relation of Free Immunoglobulin Ligh Chain Concentration to Overflow Proteinuria

Abstract Background Free immunoglobulin light chains are small proteins that undergo substantial glomerular filtration. When high serum concentrations of free light chains occur in multiple myeloma, the high filtered load of light chains exceeds the uptake capacity of proximal tubules, and overflow proteinuria ensues. Massive excretion of light chains can lead to cast formation and renal injury. The present study sought to determine the relation of serum light chain concentration to overflow proteinuria. Methods Results for patients with plasma cell disorders were reviewed as approved by an institutional review board. A Binding Site Optilite analyzer measured serum free light chain concentrations. Urine and serum protein electrophoresis were performed on Helena agarose gels. A total of 170 paired serum and 24-hour urine specimens were evaluated for 36 patients with increased kappa light chains and 26 patients with increased lambda light chains. Results Overflow proteinuria with either immunoglobulin light chain presented with light chain as the major urinary protein and limited increases of urinary albumin. Light chain excretion exceeded albumin excretion when light chain excretion surpassed 100 mg/d. Similar results were obtained for cases with increased kappa and lambda free light chains. There was a general relation of increased serum kappa and lambda light chains and urinary excretion of the corresponding light chain. Limited urinary excretion of light chains (<120 mg/d) was seen when the serum light chain concentration was < 250 mg/L, representing an approximate threshold for substantial overflow proteinuria. Overflow proteinuria exceeding 2,000 g/d was seen only when serum free light chain concentrations exceeded 800 mg/L. However, a few patients with high serum light chain values (>1,000/L) had low urinary light chain excretion that was discordant from the relation of serum light chain concentration to light chain excretion. Serum light chain values >4,000 mg/L (N = 10) were all associated with overflow proteinuria of > 1,500 mg/d and, in 7 cases, >10,000 mg/d. In a few cases, there was evidence that serum light chain assays overestimated light chain concentrations. In one case, the serum free light chain value was twice the total protein and about 12-fold higher than the serum M-spike, suggestive of substantial overestimation. Conclusions Defining the relation between serum free light chain concentrations and the magnitude of overflow proteinuria helps identify patients at low and high risk of massive overflow proteinuria that can cause renal injury. Limited overflow proteinuria (<120 mg/d) occurred when serum light chain concentration was < 250 mg/L, and substantial overflow proteinuria occurred when serum light chains exceeded 4,000 mg/L Serum free light chain concentrations above 250 mg/L were incompletely predictive of urinary free light chain excretion; a few discordant cases with high measured serum light chain concentrations (>1,000 mg/L) and low excretion of light chains were observed. Further investigation is indicated for cases with possible overestimation of free light chains or discordance between serum light chain values and urinary light chain excretion. Unexpected results in some cases could relate to polymerization of free light chains as previously reported.

Distribution of quetiapine between serum and whole blood in therapeutic drug-monitoring specimens.

Quetiapine use is on the rise, leading to a corresponding increase in acute intoxications, some of which have fatal outcomes. When assessing whole-blood quetiapine concentrations during forensic autopsies, interpretations are primarily based on toxicity data from studies of serum concentrations. To our knowledge, there are only two previous studies that have attempted to establish the ratio between whole blood and serum quetiapine concentrations with limited populations and high variability of results. Paired specimens of whole blood and serum from 16 quetiapine users recruited from the Psychiatric Clinic, St. Olav University Hospital were analyzed using LC-MS-MS. Quetiapine concentrations in both matrices were determined and compared. The mean blood:serum ratio of quetiapine was 0.74 (standard deviation (SD) = 0.05, 95% confidence interval (CI) 0.71-0.76, P < 0.001), range 0.66-0.85. Simple linear regression showed strong linear correlation between quetiapine concentrations in the two matrices (B = 0.774, P > 0.001, r = 0.999). Our results imply that quetiapine occurs at lower concentrations within erythrocytes than in plasma. This is most likely due to a high degree of plasma protein binding. Other factors which may influence the distribution of quetiapine between these compartments are solubility, metabolism and passive or active efflux mechanisms. We did not observe any covariation between blood:serum ratios and serum concentrations. Quetiapine was consistently present at lower concentrations in whole blood than in serum. If so inclined to, a conversion factor of ∼0.7 may be considered for extrapolation of concentrations from serum to whole blood, at least in cases with therapeutic quetiapine concentration levels.

B-072 Analysis of Anti-SARS-Cov-2 S1-RBD and N IgG in Saliva and Serum

Abstract Background Mucosal antibody testing may be a significant and more practical tool for measuring humoral immunity since it is non-invasive. The aim of this study was to evaluate the SARS-CoV-2-specific antibody prevalence in saliva compared to serum in 2022. Methods From February to December 2022, the paired serum and saliva specimens were collected twice over a period. The saliva specimens were collected using a Salivette Cortisol (Sarstedt AG &amp; Co. KG, Germany) device according to manufacturers’ instructions. Antibody tests were performed using nucleocapsid protein-based Elecsys anti-SARS-CoV-2 (anti-N IgG) and spike protein targeted SARS-CoV-2 IgG II Quant (anti-S1-RBD IgG). Results 162 (97.6%) of 166 had anti-S1-RBD IgG in their serum. At the 1st visit and 2nd visit, 79 (47.5%) and 97 (58.4%) patients, respectively, had anti-N IgG in their serum. At 1st visit and 2nd visit, respectively, 14 (8.4%) and 13 (7.8%) patients had anti-S1-RBD IgG in their saliva. Four (2.4%) and five (3.0%) patients had anti-N IgG in their saliva during the 1st visit and 2nd visit, respectively. The correlation coefficient of the results of anti-S1-RBD IgG and anti-N IgG quantitative results between at 1st visit and 2nd visit were low (r = 0.1721 to 0.6448). All patients in the anti-S1-RBD IgG seronegative group (N = 4) had anti-N IgG in their serum, but neither anti-S1-RBD IgG nor anti-N IgG were detected in their saliva. All anti-N IgG seronegative patients (N = 68) had anti-S1-RBD IgG in serum, while 3 patients had anti-S1-RBD IgG and 7 patients had anti-N IgG in saliva. Conclusion Mucosal antibodies were detected in certain seronegative patients. The assessment of SARS-CoV-2-specific saliva antibodies should be regarded as a supplemental non-invasive diagnostic to serum to evaluate the prevalence and transmission of COVID-19.

Intestinal microbiota links to allograft stability after lung transplantation: a prospective cohort study

Whether the alternated microbiota in the gut contribute to the risk of allograft rejection (AR) and pulmonary infection (PI) in the setting of lung transplant recipients (LTRs) remains unexplored. A prospective multicenter cohort of LTRs was identified in the four lung transplant centers. Paired fecal and serum specimens were collected and divided into AR, PI, and event-free (EF) groups according to the diagnosis at sampling. Fecal samples were determined by metagenomic sequencing. And metabolites and cytokines were detected in the paired serum to analyze the potential effect of the altered microbiota community. In total, we analyzed 146 paired samples (AR = 25, PI = 43, and EF = 78). Notably, we found that the gut microbiome of AR followed a major depletion pattern with decreased 487 species and compositional diversity. Further multi-omics analysis showed depleted serum metabolites and increased inflammatory cytokines in AR and PI. Bacteroides uniformis, which declined in AR (2.4% vs 0.6%) and was negatively associated with serum IL-1β and IL-12, was identified as a driven specie in the network of gut microbiome of EF. Functionally, the EF specimens were abundant in probiotics related to mannose and cationic antimicrobial peptide metabolism. Furthermore, a support-vector machine classifier based on microbiome, metabolome, and clinical parameters highly predicted AR (AUPRC = 0.801) and PI (AUPRC = 0.855), whereby the microbiome dataset showed a particularly high diagnostic power. In conclusion, a disruptive gut microbiota showed a significant association with allograft rejection and infection and with systemic cytokines and metabolites in LTRs.

A164 IDENTIFYING THE MOST IMPORTANT PREDICTORS TO CORRELATE SERUM METABOLITES WITH MRE CHANGES IN PATIENTS WITH PEDIATRIC CROHN DISEASE

Abstract Background Endoscopy has been the gold standard for assessing activity in Pediatric Crohn disease (pCD); however, it is limited by its invasiveness and partial assessment of small intestine and transmural inflammation. To that end, the Pediatric Inflammatory Crohn's MRE Index (PICMI) is a valid, reliable, non-invasive, and responsive index that includes transmural inflammation when assessing disease activity. The pathogenesis of pCD remains poorly understood, but evidence suggests that endogenous metabolites produced in the intestinal tract might mediate pathogenesis. Despite the important applicability of metabolomics in increasing the understanding of pCD, there has been limited research on this topic. Purpose Serum metabolomic profiles are linked to disease activity in pediatric Crohn disease. Method ImageKids is a multicenter, prospective, observational cohort study, designed to develop PICMI for pCD. The study was conducted over 18 months with paired serum specimens collected at study initiation and completion for 56 pCD patients. Due to the long time between the visits and the fact that during the study variables that highly affect serum metabolites were not controlled, we considered each patient visit as an individual measure point. Metabolites were identified using a quantitative metabolomics approach through The Metabolomics Innovation Centre (TMIC; University of Alberta). Disease activity was determined by the cutoff values in the total PICMI score of each patient. The most relevant serum metabolites were identified by medium-level and high-level variable selection analysis. Pearson correlation and hypothesis testing were used to select important metabolites. Decision trees, regularization techniques, and support vector machines were used to assess explicit importance of metabolites in disease activity. Result(s) This work provides a strategy to reduce a dimensional dataset from a metabolomic experiment. By medium-level selection analysis we were able to identify 117 statistical important metabolites for disease activity. The high-level selection analysis allowed to indicate the importance of the top 10 metabolites trough disease activity (defined by PICMI index). Results, also show that the evaluation of importance of metabolites through multivariate statistical models is dependent of the intrinsic variable selection model. Figure 1 reveals that Tryptophan ranked highest in the feature importance scoring. Histidine, Methylhistidine, Citric acid, Isoleucine, and Decanoylcarnitine also correlated well with disease severity. Image Conclusion(s) This work uses a unique approach of multivariate statistical analyses, to identify metabolites associated with pCD disease activity. Tryptophan has been previously identified as significantly altered in the blood of IBD patients compared to controls. Histidine is known to be involved in the mediation of oxidative stress, potentially influencing intestinal inflammation. These metabolites could serve as biomarkers and help define pCD pathogenesis. Disclosure of Interest None Declared

Association of CSF and Serum Neurofilament Light and Glial Fibrillary Acidic Protein, Injury Severity, and Outcome in Spinal Cord Injury.

Traumatic spinal cord injury (SCI) is highly heterogeneous, and tools to better delineate pathophysiology and recovery are needed. Our objective was to profile the response of 2 biomarkers, neurofilament light (NF-L) and glial fibrillary acidic protein (GFAP), in the serum and CSF of patients with acute SCI to evaluate their ability to objectively characterize injury severity and predict neurologic recovery. Blood and CSF samples were obtained from prospectively enrolled patients with acute SCI through days 1-4 postinjury, and the concentration of NF-L and GFAP was quantified using Simoa technology. Neurologic assessments defined the ASIA Impairment Scale (AIS) grade and motor score (MS) at presentation and 6 months postinjury. One hundred eighteen patients with acute SCI (78 AIS A, 20 AIS B, and 20 AIS C) were enrolled, with 113 (96%) completing 6-month follow-up. NF-L and GFAP levels were strongly associated between paired serum and CSF specimens, were both increased with injury severity, and distinguished among baseline AIS grades. Serum NF-L and GFAP were significantly (p = 0.02 to <0.0001) higher in AIS A patients who did not improve at 6 months, predicting AIS grade conversion with a sensitivity and specificity (95% CI) of 76% (61, 87) and 77% (55, 92) using NF-L and 72% (57, 84) and 77% (55, 92) using GFAP at 72 hours, respectively. Independent of clinical baseline assessment, a serum NF-L threshold of 170 pg/mL at 72 hours predicted those patients who would be classified as motor complete (AIS A/B) compared with motor incomplete (AIS C/D) at 6 months with a sensitivity of 87% (76, 94) and specificity of 84% (69, 94); a serum GFAP threshold of 13,180 pg/mL at 72 hours yielded a sensitivity of 90% (80, 96) and specificity of 84% (69, 94). The potential for NF-L and GFAP to classify injury severity and predict outcome after acute SCI will be useful for patient stratification and prognostication in clinical trials and inform communication of prognosis. This study provides Class I evidence that higher serum NF-L and GFAP are associated with worse neurological outcome after acute SCI. Registered on ClinicalTrials.gov: NCT00135278 (March 2006) and NCT01279811 (January 2012).

Examination of Common Coronavirus Antibodies in SARS-CoV-2-Infected and Uninfected Participants in a Household Transmission Investigation.

We compared paired serum specimens from household contacts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases with detectable SARS-CoV-2 seroconversion with contacts who remained seronegative. No protection from SARS-CoV-2 infection was associated with human coronavirus antibodies; however, an increase in common betacoronavirus antibodies was associated with seroconversion to SARS-CoV-2 in mild to moderately ill cases.

Evaluation of serological assays for SARS-CoV-2 antibody testing from dried blood spots collected from cohorts with prior SARS-CoV-2 infection

BackgroundDried blood spot (DBS) specimens are a useful serosurveillance tool particularly in hard-to-reach populations but their application for detecting SARS-CoV-2 infection is poorly characterised. ObjectivesTo compare detection of naturally acquired SARS-CoV-2 antibodies in paired DBS and serum specimens using commercially available serological immunoassays. Study DesignSpecimens were collected through St Vincent's Hospital observational post COVID-19 cohort study (ADAPT). Laboratory spotted DBS from venepuncture were initially tested on seven assays, a DBS validation completed on three with clinically collected fingerstick DBSs tested on one. ResultsSensitivity for Euroimmun nucleocapsid (NCP) IgG ELISA from laboratory spotted DBS (n=145), Euroimmun spike, IgG ELISA from laboratory spotted DBS (n=161), and Binding Site total antibody ELISA from clinically collected fingerstick DBS (n=391) was 100% (95% CI: 95.8-100%), 100% (95% CI: 95.8-100%) and 92.9% (95% CI: 89.5-95.5%), respectively. Specificity was 66.2% (95% CI: 53.6-77.0%), 96% (95% CI: 88.7-99.1%) and 98.8% (95% CI: 93.3-99.9%), respectively. All three assays’ results displayed a strong positive correlation between DBS compared to paired serum. ConclusionsThe Binding Site™ spike total antibody and Euroimmun™ spike IgG ELISAs provided good analytical performance, demonstrating that DBS specimens could facilitate specimen collection in the epidemiological surveillance of SARS-CoV-2 infection. This is highly applicable in populations and settings where venepuncture is problematic (including community based regional/remote settings, nursing homes, prisons, and schools).

Ultrasensitive detection of salivary SARS-CoV-2 IgG antibodies in individuals with natural and COVID-19 vaccine-induced immunity

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3–97.2%) compared to 91.7% (95% CI 77.5–98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3–100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AIpost/pre). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgGpost/pre). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay.

Elevated Lactate Dehydrogenase Concentrations in Plasma Compared to Serum.

To evaluate the difference in lactate dehydrogenase (LDH) concentrations in plasma vs serum specimens in our patient population. We measured LDH in 110 paired plasma and serum specimens over a 2-week period. Hemolytic indices were performed on each specimen. These paired specimens were drawn in a single setting and stored under the same conditions. For the last 14 paired specimens, cell counts were performed on the plasma/serum. Plasma LDH was on average 22% higher than serum LDH. There was no difference in the hemolytic indices between the plasma and the serum specimens. In the last 14 specimens, cell counts revealed increased platelets in the plasma specimens compared to the serum specimens. We propose switching back to using serum for LDH testing because there was unpredictable elevation in plasma LDH concentrations. These elevations in LDH levels may be linked to the platelets present in plasma and that may lyse or become activated with storage at refrigerated temperature.

A176 UTILITY OF MACHINE LEARNING FOR SERUM METABOLOMIC DATA ANALYSIS IN PEDIATRIC CROHN DISEASE

BackgroundThe pathogenesis of pCD remains poorly understood, but evidence suggests roles for genetics, environment, immune response, and gut microbes. Microbial changes can contribute to chronic inflammation and correlate with disease severity. Metabolomics reflects interactions between host immune and gut microbial function by quantifying compounds in biological samples. Therefore, metabolomics provides a unique opportunity to gain insight into pCD pathogenesis.AimsTo correlate disease severity, metabolites, and clinical data by applying machine learning algorithms in pediatric Crohn Disease (pCD).MethodsImageKids is a multicenter, prospective, cohort observational study, conducted to develop magnetic resonance enterography (MRE) indices for pCD. Paired serum specimens were collected at study initiation (Visit One; V1) and completion (Visit Four; V4; 18 months) for 120 pCD patients. Serum from patients with representative clinical scenarios and paired samples was analyzed at The Metabolomics Innovation Centre (TMIC; University of Alberta) and 131 metabolites were identified. Metabolites were analyzed via Unsupervised (U.ML) and Supervised (S.ML) Machine Learning algorithms based on Scikit-learn library in Python. Principal Component Analysis (PCA) was used to identify the variation pattern of the patients’ metabolome. Classifiers and regression algorithms were trained to assess correlation with disease activity.ResultsResults were available for the 56 paired samples. U.ML demonstrated distinct metabolome profiles with V1 clustering mainly attributed to aspartic acid, glutamic acid, and kynurenine. V4 clustering was mainly attributed to spermidine, spermine, total dimethylarginine. Furthermore, demographics was found as an important environmental factor driving distinct patterns of the metabolomics profile.After training different classifiers and regressors with S. ML algorithms, metabolome data were correlated with disease severity (defined by C-reactive protein and fecal calprotectin). Isoleucine, p-hydroxyhippuric acid, and putrescine were the top three compounds associated with disease severity. The accuracy of our classification models was of 80% and the coefficient of determination of our regression models was 0.5ConclusionsMetabolomic analysis can provide insight into disease pathogenesis and help predict disease severity among pCD patients. The correlation between metabolomics and disease severity might allow a better understanding of changes in host-microbe interactions and introduce new diagnostic or therapeutic options.Funding AgenciesCIHR

Validation of a multiplex flow immunoassay for detection of IgG antibodies against SARS-CoV-2 in dried blood spots.

BackgroundDried blood spots (DBS) are an established specimen type for clinical testing given their low cost, ease of collection and storage, and convenient shipping capabilities through the postal system. These attributes are complementary to the expansion of SARS-CoV-2 serologic testing, which may be used to inform community seroprevalence rates.MethodsThe Luminex xMAP SARS-CoV-2 Multi-Antigen assay utilizes magnetic beads labeled with three viral antigens (nucleocapsid [NC], receptor binding domain [RBD], spike S1 subunit) to detect anti-viral IgG-class antibodies, and has Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use in serum and plasma. This assay was modified for use with DBS and validated against paired sera tested by one of two reference assays: the Roche Diagnostics Elecsys anti-SARS-CoV-2 ECLIA or the Euroimmun anti-SARS-CoV-2 IgG ELISA.Results159 paired DBS and serum specimens analyzed using the modified Luminex xMAP assay on DBS and the reference methods on serum showed an overall concordance of 96.9% (154/159). Use of multivariate pattern recognition software (CLIR) for post-analytical interpretation of the Luminex xMAP DBS assay results, instead of manufacturer provided interpretive thresholds, increased overall qualitative result concordance to 99.4% (158/159) between the modified Luminex xMAP DBS and reference results.ConclusionsUse of DBS for detection of antibodies against SARS-CoV-2 provides comparable results to those obtained using serum. DBS concordance was improved with multivariate pattern recognition software (CLIR). We demonstrate that DBS are a reliable specimen type for SARS-CoV-2 antibody detection using the modified Luminex xMAP assay.

Urine miRNA signature as a potential non-invasive diagnostic and prognostic biomarker in cervical cancer

MicroRNAs as cancer biomarkers in serum, plasma, and other body fluids are often used but analysis of miRNA in urine is limited. We investigated the expression of selected miRNAs in the paired urine, serum, cervical scrape, and tumor tissue specimens from the women with cervical precancer and cancer with a view to identify if urine miRNAs could be used as reliable non-invasive biomarkers for an early diagnosis and prognosis of cervical cancer. Expression of three oncomiRs (miR-21, miR-199a, and miR-155-5p) and three tumor suppressors (miR-34a, miR-145, and miR-218) as selected by database search in cervical pre-cancer, cancer, and normal controls including cervical cancer cell lines were analyzed using qRT-PCR. The expression of miRNAs was correlated with various clinicopathological parameters, including HPV infection and survival outcome. We observed a significant overexpression of the oncomiRs and the downregulation of tumor suppressor miRNAs. A combination of miR-145-5p, miR-218-5p, and miR-34a-5p in urine yielded 100% sensitivity and 92.8% specificity in distinguishing precancer and cancer patients from healthy controls and it well correlates with those of serum and tumor tissues. The expression of miR-34a-5p and miR-218-5p were found to be independent prognostic factors for the overall survival of cervical cancer patients. We conclude that the evaluation of the above specific miRNA expression in non-invasive urine samples may serve as a reliable biomarker for early detection and prognosis of cervical cancer.

Detection of SARS-CoV-2 IgG antibodies in dried blood spots

The COVID-19 pandemic led to development of numerous serologic tests. To obviate the need for phlebotomy services, we validated dried blood spots (DBS) for anti-SARS-CoV-2 serologic testing. Immunoglobulins were extracted from 3 mm DBS punches and the extracts were analyzed using the Euroimmun anti-SARS-CoV-2 IgG ELISA. Various pre-analytical factors were studied. Results were favorable for DBS stored for at least 67 days at or below 37°C. Comparison between paired serum and DBS specimens tested by the Euroimmun ELISA showed 96.8% and 81.3% positive and negative agreement, respectively, indicating that confirmatory testing of positive Euroimmun results on DBS extracts is necessary to achieve clinical accuracy. Our findings suggest that any SARS-CoV-2 antibody assay that requires pre-dilution of serum is amenable to DBS as an alternate specimen type that is relatively low cost, self-collectable, stable, can be shipped by standard mail and could be used to establish the seroprevalence of large populations.

Rickettsial Infections among the Undifferentiated Febrile Patients Attending a Tertiary Care Teaching Hospital of Northern India: A Longitudinal Study.

BackgroundAcute undifferentiated febrile illness (AUFI) is one of the most daunting challenges a physician faces in such settings. Among AUFI, rickettsial infections are most common and related infections (such as anaplasmosis, ehrlichiosis, and Q fever) which are caused by an unusual type of bacteria that can live only inside the cells of another organism. The present study was therefore planned with an objective to estimate the prevalence of rickettsial infection among patients of undifferentiated fever and to determine any association of socio-demographic characteristics with rickettsial disease.Materials and MethodsPatients presenting with febrile illness and admitted or attending out-patient department of Sher-i-Kashmir Institute of Medical Sciences, Srinagar was approached and recruited in the study. Weil Felix Assay, enzyme-linked immunosorbent assay and indirect immunofluorescence assay were done to detect the anti-rickettsial antibodies. Serological evidence of a fourfold increase in IgG-specific antibody titer reactive with spotted fever group rickettsial antigen by indirect immunofluorescence antibody assays between paired serum specimens was considered a confirmatory diagnosis for the rickettsial disease.ResultsMost of the patients were males 61.6%, and most 46.2% were in the age group of 20 -39 years. Most of the patients, 80.8% belonged to rural areas, and 48% belonged to the upper middle (II) class of the socio-economic class according to modified Kuppuswamy scale. Of the studied participants, a majority, 47.0%, were determined undiagnosed, while 15.4% studied participants were diagnosed to have a rickettsial disease. In patients positive for typhus group, 67.8% were IgM positive, 28.5% were IgG positive, and only 3% were positive for IgM and IgG. In patients positive for Scrub Typhus Group, 32.7% were positive for IgM, and 62.0% were positive for IgG, and only 5.0% were positive for both IgM and IgG. In patients positive for spotted fever group, 36.1% were positive for IgM, and 58.5% were positive for IgG, and only 5.5% were positive for both IgM and IgG. The prevalence of rickettsial disease was found to be 11.3%.ConclusionRickettsial diseases, typhoid and brucellosis, were the most prevalent diseased diagnosed among patients reporting to hospitals with undifferentiated febrile illness. Clinicians must consider rickettsial diseases as one of the differential diagnosis while treating patients with fever.

Concomitant Imbalances of Systemic and Mucosal Immunity Increase HIV Acquisition Risk

We previously reported association of increased cervical RANTES and decreased secretory leukocyte protease inhibitor (SLPI) with higher risk of HIV acquisition in reproductive-age women. We now examine the interaction of concomitantly altered systemic and cervical immunity on such risk. We measured immune biomarkers in 4390 cervical and 2390 paired serum specimens at quarterly visits in 218 HIV seroconverters and 784 seronegative women. We assessed proinflammatory (IL-1β, IL-6, IL-8, MIP-3α, and RANTES), anti-inflammatory (IL-1RA and SLPI), vascular activation (vascular endothelial growth factor and Intercellular Adhesion Molecule-1) and defensin (BD2) cervical biomarkers and systemic (peripheral blood) C reactive protein (CRP), IL-6, IL-7, and sCD14 as indicators of immune dysregulation. Biomarker levels were Box-Cox transformed and odds ratios for HIV acquisition calculated based on top quartile or higher/lower than median levels for all HIV-negative visits. Subsequent HIV acquisition was associated with 5 of 14 individual biomarkers: low systemic CRP [odds ratio (OR) = 1.49, 1.21-1.83] and IL-6 (OR = 1.23, 1.00-1.51), high cervical BD-2 (OR = 1.33, 1.11-1.58) and RANTES (OR = 1.20, 1.01-1.43), and low cervical IL-1RA (OR = 0.65, 0.48-0.86). Low systemic CRP concomitant with altered cervical immunity, especially high BD2, conveyed highest HIV risk (1.63, 1.29-2.05). Additional markers of increased risk emerged when low systemic CRP coincided with: low systemic IL-6 and IL-7 (OR = 1.53, 1.18-1.97); high cervical IL-8 and MIP-3α (OR = 1.40, 1.07-1.83); high cervical IL-1β and IL-6 (OR = 1.43, 1.09-1.86); or low cervical SLPI (OR = 1.36, 1.08-1.71). Changes in both peripheral and mucosal immunity may precede and predispose women to HIV infection. Suppressed systemic immunity (ie, low CRP) alone or in combination with imbalanced cervical innate immunity (high proinflammatory and low anti-inflammatory mediators) indicated increased vulnerability to infection. Understanding these combined effects on HIV susceptibility is essential to preventing new infections.

2025. Procalcitonin Reference Cutoff and Specimen-Type Validation

InvestigationThe primary objective of this study was to validate that the clinical cutoff of 0.3 ng/mL is appropriate in our population of patients. The secondary objective of this study was to compare procalcitonin results in plasma and serum specimens to prove that both sample types gave similar results.BackgroundOur institution started performing procalcitonin testing on November 29, 2017. The Roche package insert indicated that in a population of 282 self-reported healthy individuals, the 95th percentile, upper reference range limit was calculated at 0.08 ng/mL. This 95th percentile cutoff seemed to be much lower than published clinical cutoffs of 0.25 and 0.30 ng/mL. Upon extensive literature review and discussion with pediatric and adult infectious disease physicians, we decided to use the clinical cutoff of 0.3 ng/mL to indicate invasive bacterial infection in our institution.MethodsTwo hundred and fifty patients with normal renal and hepatic function, white blood cell counts and C-reactive protein below 5.0 mg/L were selected for the validation of procalcitonin clinical cutoff. An additional 100 patients with paired plasma and serum specimens drawn at the same time were selected for the plasma vs. serum comparison study. All results were analyzed using the R statistical program.ResultsBlood specimens from 128 females and 122 male patients ranging from 10 to 84 years old were utilized in this study. Out of the 250 specimens, 128 were plasma and 122 were serum specimens. The procalcitonin 95th percentile upper reference range limit based on our population was 0.08 ng/mL, which validated the Roche package insert claim. Only one out of 250 patients (0.4%) had procalcitonin result above 0.3 ng/mL. The paired plasma and serum specimens in 100 patients yielded procalcitonin results that correlated with a Pearson’s R value of 0.999. The linear regression equation for the correlation was y = 0.94x.ConclusionThis result confirmed that a procalcitonin clinical cutoff of 0.3 ng/mL is appropriate in our patient population. It also showed that both plasma and serum specimens can be used for procalcitonin measurement.Disclosures M. Ahmad, Roche: Collaborator, Research support. A. Adaya, Roche: Collaborator, Research support. D. Patel, Roche: Collaborator, Research support. R. Benirschke, Roche: Collaborator, Research support. H. Lee, Roche: Collaborator, Research support.

Development and Use of an Endpoint Titration Assay To Characterize Mumps IgG Avidity following Measles, Mumps, and Rubella Vaccination and Wild-Type Mumps Infection.

Waning mumps IgG antibody and incomplete IgG avidity maturation may increase susceptibility to mumps virus infection in some vaccinees. To measure mumps IgG avidity, serum specimens serially diluted to the endpoint were incubated on a commercial mumps-specific IgG enzyme immunoassay and treated with the protein denaturant diethylamine (60 mM, pH 10). End titer avidity indices (etAIs [percent ratio of detected diethylamine-resistant IgG at endpoint]) were calculated. Unpaired serum specimens (n = 108) from 15-month-old children living in a low-incidence setting were collected 1 month and 2 years after the first measles, mumps, and rubella vaccine dose (MMR1) and tested for mumps avidity. Per the receiver operating characteristic curve, the avidity assay is accurate (area under the curve, 0.994; 95% confidence interval [CI], 0.956 to 1.000), 96.5% sensitive (95% CI, 87.9 to 99.6%), and 92.2% specific (95% CI, 81.1 to 97.8%) at an etAI of 30%. When 9 sets of paired serum specimens collected 1 to 60 months post-MMR1 were tested for mumps and measles IgG avidity using comparable methods, the mumps etAI increased from 11% to 40 to 60% in 6 months. From 6 to 60 months, avidity was sustained at a mean etAI of 50% (95% CI, 46 to 54%), significantly lower (P < 0.0001) than the mean measles etAI of 80% (95% CI, 74 to 86%). Mean etAIs in children 2 years post-MMR1 (n = 51), unvaccinated adults with distant mumps disease (n = 29), and confirmed mumps cases (n = 23) were 54, 62, and 57%, respectively. A mumps-specific endpoint avidity assay was developed and validated, and mumps avidity was determined to be generally sustained at etAIs of 40 to 60%, reaching etAIs of >80% in some individuals.IMPORTANCE Numerous outbreaks of mumps have occurred in the United States among two-dose measles-mumps-rubella (MMR)-vaccinated populations since 2006. The avidity of mumps-specific IgG antibodies may affect susceptibility to mumps virus infection in some vaccinated individuals. To accurately measure mumps avidity, we developed and validated a mumps-specific IgG avidity assay that determines avidity at the endpoint titer of serially diluted serum specimens, providing results that are independent of IgG concentration. At low antibody titers, endpoint methods are considered more accurate than methods that determine avidity at a single dilution. We determined that 6 months after the first MMR dose, mumps IgG avidity is high and generally sustained at avidity indices of 40 to 60%, reaching values of >80% in some individuals. Additionally, 4% (4/103) of individuals had avidity indices of ≤30% (low avidity) 2 years after vaccination. Inadequate mumps avidity maturation may be one factor influencing susceptibility to mumps virus infection among previously vaccinated or naturally infected individuals.

Intrathecal K free light chain synthesis in multiple sclerosis at clinical onset associates with local IgG production and improves the diagnostic value of cerebrospinal fluid examination

Background. The pathological significance and the diagnostic usefulness of intrathecal κ and λ free light chain (FLC) synthesis in Multiple Sclerosis (MS) are debated. Methods. Paired cerebrospinal fluid (CSF) and serum specimens from 70 relapsing remitting MS (RRMS), 40 with and 30 without CSF restricted IgG Oligoclonal Band (IgGOB), and 37 from healthy controls (HC) were analyzed. IgG, IgM, κFLC and λFLC concentrations and indexes were evaluated. All RRMS performed MRI to estimate white and grey matter (WM) pathology. Results. In HC, no intrathecal κ or λ FLC synthesis was found, and κFLC and λFLC Indexes were reciprocally correlated (r = 0.67, p < 0.001). In RRMS, intrathecal κFLC or λFLC synthesis was demonstrated in respectively 66% and 43% of the cases, the Qκ/λ ratio was significantly higher compared to HC (17.0 ± 31.3 vs 0.79 ± 0.20, p < 0.001) and the correlation between κFLC Index and λFLC Index was weak (r:0.38, p < 0.05). Intrathecal IgG synthesis was associated with κFLC Index (IgG Index: r2 = 0.53, β = 0.73, p < 0.001; IgGLOC: r2 = 0.37, β = 0.61, p < 0.001; IgGIF: r2 = 0.69, β = 0.83, p < 0.001), but not with λFLC Index, while intrathecal IgM synthesis correlated with λFLC Index (IgM Index: r = 0.41, p < 0.001; IgMLOC: r = 0.34, p < 0.005; IgMIF: r = 0.45, p < 0.001), but not with κFLC Index. 26% of RRMS patients without CSF-restricted IgGOB had increased κFLCLOC. Finally, no associations were observed between any CSF and MRI parameters. Conclusions. The demonstration of intrathecal κFLC synthesis may further improve the diagnostic usefulness of CSF examination in RRMS. The marked increased in Qκ/λ further suggests a deregulated B-cell activation in MS pathology.