Comparison of the time required for manual (visually read) and semi-automated POCT urinalysis and pregnancy testing with associated electronic medical record (EMR) transcription errors.

BACKGROUND: POCT urinalysis (UA) and urine pregnancy tests (UPT) are routinely performed in obstetrics and gynecology (Ob/Gyn) clinics by dipstick and pregnancy test kit methods respectively. In this study, we compared the time, efficiency and accuracy of these tests using manual, visually read methods and a semi-automated analyzer that was not interfaced to the EMR. METHODS: We prospectively enrolled 2525 patients at five Ob/Gyn clinics. Urine samples were tested using three different dipsticks for UA (2, 7 and 10 test pads) and the Sure-Vue™ urine pregnancy test kit. The samples were analyzed on the CLINITEK Status® Connect System and results compared for time taken and errors in results' transcription. RESULTS: Using the CLINITEK Status Connect System, average test time and average total test time for UA dipsticks 7 and 10 test pads was significantly less than the manual, visually read method (0.77 and 0.64 min, respectively; p < 0.001). The average test time for manual, visually read Chem 2 was significantly less than the CLINITEK Status Connect System (0.09 min; p = 0.005), but not the average total test time (0.08 min; p = 0.33). Average test time for a negative UPT using the CLINITEK Status Connect System was significantly greater (0.87 min; p < 0.001). We found a transcription error rate of 0.3-1.7% for UA results and none for UPT. About 8% of UA and 12% of UPT results were not documented in EMR. CONCLUSION: The CLINITEK Status Connect System was more efficient than the manual, visually read process and if interfaced with the EMR would eliminate errors and non-documentation of results.

Critically low sodium levels due to concentration gradients formed in patient samples after undergoing a freeze-thaw cycle.

BACKGROUND: Concentrations gradients that form in plasma as a result of freezing and thawing is a well-known phenomenon. As the water fraction converts into ice, plasma constituents diffuse from the freezing front by natural convection in the liquid phase. This process can lead to erroneous lab results, if the sample is not thoroughly mixed prior to testing. METHODS: A series of patient samples received at the clinical chemistry core lab were found to have low sodium levels that normalized after tube inversion. We suspected that the samples may have frozen during shipping and therefore examined the effects of freezing and thawing on serum. RESULTS: Our investigation revealed that prior to arriving at the core lab, samples from one of our satellite clinics were undergoing a freeze-thaw cycle during shipping, which resulted in the formation of concentration gradients and spurious lab results on arrival. CONCLUSIONS: Large hospitals that have a central core lab and receive patient samples from satellite clinics need to be aware of this phenomena, which can contribute to erroneous lab results being posted in a patient's electronic medical record, resulting in a misdiagnosis.

What's a Lab to Do During and After a Hurricane?

Although laboratories may be able to rely on a comprehensive Hurricane Plan during a hurricane, alarming and unanticipated events frequently occur. To minimize disruption of lab operations, it is important to try to mitigate the impact of these unexpected events as quickly as possible, in the quest to minimize negative outcomes. In this article, we discuss approaches to dealing with unanticipated events during and after hurricanes, based on our personal experiences.

Comparison of the Alere i Strep A Test and the BD Veritor System in the Detection of Group A Streptococcus and the Hypothetical Impact of Results on Antibiotic Utilization.

Rapid detection of group A Streptococcus (GAS) is an integral component of treatment decisions in the clinic, especially in the pediatric population. We prospectively collected 216 specimens from symptomatic, predominantly pediatric patients and evaluated the performance of the Alere i Strep A test (Alere i; Alere Inc., Scarborough, ME) and the BD Veritor system (BD Veritor; Becton, Dickinson and Company, Sparks, MD), with culture results being used as a comparator. Real-time PCR (RT-PCR) was performed as an arbiter in discordant cases. Comprehensive chart review was also done to determine the hypothetical impact of the results on antibiotic use. Alere i had a sensitivity and a specificity of 100% and 91.3%, respectively, and BD Veritor had a sensitivity and a specificity of 76.2% and 93.6%, respectively, when the results were compared to those of GAS culture. Further analysis of discordant results using RT-PCR revealed that while BD Veritor missed 13 confirmed positive cases, Alere i detected 100% (n = 13) of the same cases. Analysis of assay agreement showed that Alere i and BD Veritor had only moderate agreement (agreement = 0.888 [95% confidence interval {CI}, 0.838 to 0.927]; kappa index = 0.689 [95% CI, 0.91 to 0.974]). We also found both the underutilization and the overutilization of antibiotics based on the results of molecular testing. Overall, Alere i showed superior performance over BD Veritor in the detection of GAS pharyngitis and could potentially assist in better antibiotic utilization.

Data documenting the performance of the PT/INR line correction method for reconciling INR discrepancies between central laboratory coagulation analyzers using different thromboplastins during the evaluation of a portable Coagulometer.

The data presented here was produced as part of an evaluation of the performance of the CoaguChek XS point-of-care coagulation analyzer, which is discussed in the research article "POCT PT INR - Is it adequate for Patient Care? A Comparison of the Roche Coaguchek XS vs. Stago Star vs. Siemens BCS in Patients Routinely Seen in an Anticoagulation Clinic" (Baker et al., in press) [1]. An effort to reconcile discrepancies in the patient INR result distributions from different central lab instruments (Stago Star and Siemens BCS) with the PT/INR line method is described (Poller et al., 2010, 2011; Ibrahim et al., 2011) [2], [3], [4]. While regression analysis of the ECAA Poller calibrant data provided a linear PT/INR line for all methods, Pearson's chi-squared and one-way repeated measures ANOVA analyses showed that central lab INR measurements continued to exhibit measurement site dependence after the PT/INR line correction was applied. According to paired t-test analysis, only the human thromboplastin dependent methods (CoaguChek XS and Siemens BCS both before and after PT/INR line correction) showed statistically significant agreement (p-value >0.05).

POCT PT INR - Is it adequate for patient care? A comparison of the Roche Coaguchek XS vs. Stago Star vs. Siemens BCS in patients routinely seen in an anticoagulation clinic.

BACKGROUND: In this study we examined the difference in patient INR values as measured by the POCT CoaguChek XS device and central laboratory Stago Evolution and Siemens BCS XP analyzers. METHODS: This study composed of 100 warfarin therapy patients and 20 coagulation normal subjects, showed that the difference between the POCT and clinical laboratory values increased with increasing INR and was exacerbated by the use of different thromboplastin reagents by the POCT and central lab. RESULTS: The CoaguChek XS and on-site Stago analyzers which used human recombinant (ISI=1.01) and rabbit brain thromboplastin (ISI=1.25), respectively, showed reasonable agreement for INR<3.0 (k=0.62) but significant difference for INR≥3.0 (k=0.10). In contrast, the CoaguChek XS and Siemens BCS XP, which both employed human recombinant thromboplastin (BCS ISI=1.02), showed greater agreement for the complete range INR values (INR<3.0 k=0.84; INR≥3.0 k=0.70). ECAA Poller calibrant data showed the automated instruments were performing as expected, indicating that ISI calibrations were correct but insufficient to standardize the INR values for the different thromboplastin methods across the full range of measured INRs. Central lab verification of POCT INR>5.0 with the Stago Evolution prevented adverse treatment events for the warfarin therapy patients in the six months preceding and following this investigation.

Comparison of the Alere i and BD Veritor Assays for the Rapid Detection of Influenza A and B Viruses.

BACKGROUND: The use of point-of care testing (POCT) in patient management decisions is becoming increasingly common. Our goal was to evaluate the diagnostic performance of 2 commercially available rapid POCT devices for influenza viruses A and B: the Alere™ i Instrument (Alere, Scarborough) and the BD Veritor™ System (BD Diagnostics). METHODS: Paired nasopharyngeal swabs were collected from patients (18-71 years) presenting with influenza-like symptoms at 3 outpatient clinics. A total of 65 samples were obtained. The Alere i and BD Veritor were performed according to the manufacturers' instructions. Discordant results were resolved using real-time reverse transcription PCR (RT-PCR). RESULTS: In a head-to-head comparison involving symptomatic adult patients visiting outpatient clinics during the 2014-2015 and 2015-2016 influenza seasons, the Alere i and BD Veritor had 90.63% agreement in the detection of influenza A virus and a statistically significant observed κ coefficient of 0.754 (P <0.0001). Discordant results between the Alere i and BD Veritor were further investigated using RT-PCR, showing that the BD Veritor missed 5 positive influenza A virus results (false negatives) and detected 1 false positive, while the Alere i results agreed with all RT-PCR results. There were no discordant results between the Alere i and BD Veritor in the detection of influenza B virus. CONCLUSIONS: Our data suggest that the Alere i has higher sensitivity and specificity than the BD Veritor in the detection of influenza A virus. Both assays showed equal performance in the detection of influenza B virus.

S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure.

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

Skin Rash and Microscopic Hematuria in a 10-Year-Old Caucasian Male.

OBJECTIVE: Henoch-Schonlein purpura (HSP) is an acute, systemic, vasculitis with IgA-dominant immune deposits. With the emphasis on educational value of HSP, which is the most common form of vasculitis in children, we report an actual case from a 10-year-old boy. METHOD: The patient presented with the chief complaint of a skin rash. His illness history, family medical history, physical examination, and relevant laboratory findings were summarized, followed by a question and possible answer format discussion. RESULTS AND CONCLUSION: With the significant elevation of red blood cells in his urine and moderate to severe deposition of IgA in kidney biopsy, the patient was diagnosed with HSP nephritis. Renal symptoms, such as proteinuria and hematuria, are mostly the last to develop and determine the long-term prognosis in HSP patients. The patient is currently undergoing steroid treatment, which is the primary intervention for HSP as it spontaneously resolves in most of affected children.

Targeted proteomics for biomarker discovery and validation of hepatocellular carcinoma in hepatitis C infected patients.

Hepatocellular carcinoma (HCC)-related mortality is high because early detection modalities are hampered by inaccuracy, expense and inherent procedural risks. Thus there is an urgent need for minimally invasive, highly specific and sensitive biomarkers that enable early disease detection when therapeutic intervention remains practical. Successful therapeutic intervention is predicated on the ability to detect the cancer early. Similar unmet medical needs abound in most fields of medicine and require novel methodological approaches. Proteomic profiling of body fluids presents a sensitive diagnostic tool for early cancer detection. Here we describe such a strategy of comparative proteomics to identify potential serum-based biomarkers to distinguish high-risk chronic hepatitis C virus infected patients from HCC patients. In order to compensate for the extraordinary dynamic range in serum proteins, enrichment methods that compress the dynamic range without surrendering proteome complexity can help minimize the problems associated with many depletion methods. The enriched serum can be resolved using 2D-difference in-gel electrophoresis and the spots showing statistically significant changes selected for identification by liquid chromatography-tandem mass spectrometry. Subsequent quantitative verification and validation of these candidate biomarkers represent an obligatory and rate-limiting process that is greatly enabled by selected reaction monitoring (SRM). SRM is a tandem mass spectrometry method suitable for identification and quantitation of target peptides within complex mixtures independent on peptide-specific antibodies. Ultimately, multiplexed SRM and dynamic multiple reaction monitoring can be utilized for the simultaneous analysis of a biomarker panel derived from support vector machine learning approaches, which allows monitoring a specific disease state such as early HCC. Overall, this approach yields high probability biomarkers for clinical validation in large patient cohorts and represents a strategy extensible to many diseases.

A1c Gear: Laboratory quality HbA1c measurement at the point of care.

INTRODUCTION: HbA1c is an important part of assessing the diabetic control and since the use of point-of-care devices for monitoring HbA1c is increasing, it is important to determine how these devices compare to the central laboratory. METHODS: One hundred and twenty patient samples were analyzed on the Bio-Rad Variant™II and one POC analyzer (Sakae A1c Gear). Three patient sample pools containing ~5%, ~7%, and ~10% HbA1c levels were run over 20 days. Three reagent lots and three instruments were evaluated for the A1c Gear. RESULTS: The 120 patient samples showed strong correlation (R(2)>0.989) when compared to the Variant™II with means=8.06% and 7.81%, for Variant IIand A1c Gear, respectively. Changing reagent lots or instruments had no impact for the A1c Gear. The ~5%, ~7%, and ~10% pools within-run and between-run imprecision was between 0.87-1.33% and 1.03-1.32%, and 1.41-2.35% and 1.24-1.89% with total imprecision of 1.67-2.35% and 1.61-2.31% for the A1c Gear and Variant II, respectively. The A1c Gear showed a small negative bias (0.25% HbA1c) across HbA1c measurement ranges of <11.5%. This bias was, however, acceptable and not considered to be clinically significant. CONCLUSIONS: The A1c Gear meets the criteria of total CV <3% leading us to the conclusion that the A1c Gear can give results as precise as the laboratory at the POC.

Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma.

BACKGROUND: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%-80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. METHODS: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. RESULTS: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. CONCLUSIONS: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

Evaluation of the aspartate aminotransferase/platelet ratio index and enhanced liver fibrosis tests to detect significant fibrosis due to chronic hepatitis C.

BACKGROUND: The assessment of liver fibrosis in chronic hepatitis C patients is important for prognosis and making decisions regarding antiviral treatment. Although liver biopsy is considered the reference standard for assessing hepatic fibrosis in patients with chronic hepatitis C, it is invasive and associated with sampling and interobserver variability. Serum fibrosis markers have been utilized as surrogates for a liver biopsy. METHODS: We completed a prospective study of 191 patients in which blood draws and liver biopsies were performed on the same visit. Using liver biopsies the sensitivity, specificity, and negative and positive predictive values for both aspartate aminotransferase/platelet ratio index (APRI) and enhanced liver fibrosis (ELF) were determined. The patients were divided into training and validation patient sets to develop and validate a clinically useful algorithm for differentiating mild and significant fibrosis. RESULTS: The area under the ROC curve for the APRI and ELF tests for the training set was 0.865 and 0.880, respectively. The clinical sensitivity in separating mild (F0-F1) from significant fibrosis (F2-F4) was 80% and 86.0% with a clinical specificity of 86.7% and 77.8%, respectively. For the validation sets the area under the ROC curve for the APRI and ELF tests was, 0.855 and 0.780, respectively. The clinical sensitivity of the APRI and ELF tests in separating mild (F0-F1) from significant (F2-F4) fibrosis for the validation set was 90.0% and 70.0% with a clinical specificity of 73.3% and 86.7%, respectively. There were no differences between the APRI and ELF tests in distinguishing mild from significant fibrosis for either the training or validation sets (P=0.61 and 0.20, respectively). Using APRI as the primary test followed by ELF for patients in the intermediate zone, would have decreased the number of liver biopsies needed by 40% for the validation set. Overall, use of our algorithm would have decreased the number of patients who needed a liver biopsy from 95 to 24-a 74.7% reduction. CONCLUSIONS: This study has shown that the APRI and ELF tests are equally accurate in distinguishing mild from significant liver fibrosis, and combining them into a validated algorithm improves their performance in distinguishing mild from significant fibrosis.

Elevation of serum fortilin levels is specific for apoptosis and signifies cell death in vivo.

BACKGROUND: Billions of cells undergo apoptosis each day in the average normal adult. The ability to readily assess the degree of apoptosis in human diseases is hampered by the lack of sensitive and specific serum biomarkers of apoptosis. Fortilin is a novel prosurvival molecule that protects cells against various noxious stimuli. While fortilin is secreted into the extracellular space under certain conditions, the relationship between the serum concentration of fortilin and the presence and extent of apoptosis in vivo remains unknown. METHODS & RESULTS: Using a newly developed fortilin ELISA system, we show here that fortilin exists in the normal human and mouse circulation. We further demonstrate that fortilin serum levels are significantly elevated in patients with solid cancer, in response to anti-cancer chemo- or radiation therapy. The elevation of fortilin serum levels is more robust and sensitive than that of such previously-reported serum biomarkers of apoptosis as fragmented cytokeratin-18, cytochrome c, and nucleosomal DNA. In addition, targeted apoptotic liver damage induced by Jo2 anti-Fas (CD95) antibody consistently and significantly increased serum fortilin levels in C57BL/6J mice. Finally, when challenged by anti-human-Fas IgM antibody, Jurkat leukemic T cells apoptosed and released fortilin into the medium before plasma membrane integrity was compromised. CONCLUSIONS: Taken together, these data suggest that serum fortilin levels reflect the degree and extent of apoptosis occurring in vivo. GENERAL SIGNIFICANCE: Fortilin is a viable serum biomarker of in vivo apoptosis and can be utilized to noninvasively assess the status of in vivo apoptosis in humans.

Biomarker discovery for early detection of hepatocellular carcinoma in hepatitis C-infected patients.

Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of (18)O/(16)O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using (18)O/(16)O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.

Innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease.

BACKGROUND: We investigated the pathological and diagnostic role of selected markers of inflammation, oxidant/antioxidant status, and cellular injury in human Chagas disease. METHODS: Seropositive/chagasic subjects characterized as clinically-symptomatic or clinically-asymptomatic (n = 116), seronegative/cardiac subjects (n = 102), and seronegative/healthy subjects (n = 45) were analyzed for peripheral blood biomarkers. RESULTS: Seropositive/chagasic subjects exhibited an increase in sera or plasma levels of myeloperoxidase (MPO, 2.8-fold), advanced oxidation protein products (AOPP, 56%), nitrite (5.7-fold), lipid peroxides (LPO, 12-17-fold) and malondialdehyde (MDA, 4-6-fold); and a decline in superoxide dismutase (SOD, 52%) and glutathione (GSH, 75%) contents. Correlation analysis identified a significant (p<0.001) linear relationship between inflammatory markers (AOPP/nitrite: r = 0.877), inflammation and antioxidant/oxidant status (AOPP/glutathione peroxidase (GPX): r = 0.902, AOPP/GSH: r = 0.806, Nitrite/GPX: 0.773, Nitrite/LPO: 0.805, MDA/MPO: 0.718), and antioxidant/oxidant levels (GPX/MDA: r = 0.768) in chagasic subjects. Of these, MPO, LPO and nitrite biomarkers were highly specific and sensitive for distinguishing seropositive/chagasic subjects from seronegative/healthy controls (p<0.001, training and fitting AUC/ROC >0.95). The MPO (r = 0.664) and LPO (r = 0.841) levels were also correlated with clinical disease state in chagasic subjects (p<0.001). Seronegative/cardiac subjects exhibited up to 77% decline in SOD, 3-5-fold increase in LPO and glutamate pyruvate transaminase (GPT) levels, and statistically insignificant change in MPO, AOPP, MDA, GPX, GSH, and creatine kinase (CK) levels. CONCLUSIONS: The interlinked effects of innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease. The MPO, LPO and nitrite are excellent biomarkers for diagnosing seropositive/chagasic subjects, and MPO and LPO levels have potential utility in identifying clinical severity of Chagas disease.

Using immunoproteomics to identify alpha-enolase as an autoantigen in liver fibrosis.

Liver fibrosis results from extracellular matrix accumulation during the wound healing process when the liver is insulted with chronic viral infection, inflammation, or alcoholic diseases. The current diagnosis of liver fibrosis is mainly dependent on biopsy, which is an invasive approach. Identification of serological biomarkers has been considered as the most promising way for early detection of the disease. Although several biomarkers in liver fibrosis have been identified, the problem is that these markers can be also detected in fibrogenesis that occurred in other organs. In this study, we have identified and characterized some cellular proteins that can be recognized by autoantibodies in the sera from patients with precirrhotic stage of liver fibrosis. Among 180 sera from patients with liver fibrosis, 14.4% (26/180) of sera contained autoantibody against a protein migrating around 47 kDa on SDS-PAGE gel. Indirect immunofluorescence assay using purified autoantibody against the 47-kDa protein showed that this protein mainly localized in the cytoplasm. Using immunoproteomic approach, the 47-kDa protein was identified as alpha-enolase. In further study, the frequency of antialpha-enolase antibody in sera from patients with precirrhotic stage of liver fibrosis (21.6%, 27/125) was significantly higher than that in sera from patients with cirrhosis (9.1%, 5/55) and liver cancer (14.3%, 12/84), as well as in sera from healthy individuals (4.1%, 3/74). Therefore, alpha-enolase is an autoantigen that elicits autoimmune response in liver fibrosis and can be a potential prognostic factor for liver fibrosis diagnosis.

Antigenicity and diagnostic potential of vaccine candidates in human Chagas disease.

BACKGROUND: Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. METHODS AND RESULTS: Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st)-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd)-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcG(mix) were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcG(mix)- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects. CONCLUSIONS: Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.

Evaluation of the performance of urine albumin, creatinine and albumin-creatinine ratio assay on two POCT analyzers relative to a central laboratory method.

BACKGROUND: The evaluation of microalbumin, creatinine and albumin-creatinine ratio is very important in patients with diabetes for the early detection of kidney disease and the identification of patients at risk for complications from diabetes or hypertension. METHODS: A total of 88 spot urine samples previously analyzed using the Vitros 5,1 FS (creatinine) and Beckman Coulter Immage (microalbumin) located in the central laboratory and having microalbumin and creatinine values within the Afinion and DCA Vantage reportable ranges were run on 2 point of care (POC) instruments (Siemens DCA Vantage and Axis-Shield Afinion). RESULTS: The mean values for the DCA Vantage were: 42.6 mg/l for albumin, 10.3 mol/l for creatinine, and 5.4 mg/mol for ACR. For the Afinion AS100, the mean values were: 48.5mg/l for albumin, 9.5 mol/l for creatinine, and 6.7 mg/mol for ACR. The mean values obtained for CL were: 40.8 mg/l for albumin, 10.0 mol/l for creatinine, and 5.4 mg/mol for ACR. All POC analyzers showed good correlation to the central laboratory tests for microalbumin, creatinine and albumin creatinine ratio (ACR) for Afinion (R(2)=0.954, 0.974, and 0.964, respectively) and DCA Vantage (R(2)=0.989, 0.987, and 0.991, respectively). With the exception of the DCA Vantage ACR (p=0.53), the levels of microalbumin, creatinine and ACR obtained for the Afinion and DCA Vantage instruments as compared to the CL were statistically different (p<0.05). The inter and intraday imprecision for both POC instruments was <2.9% and total imprecision <8.7%. CONCLUSIONS: The 2 instruments evaluated in this study were in good agreement with the quantitative laboratory results and thus can be used for microalbumin, creatinine and ACR assays at the POC. However, facilities using Afinion will have to use different normal range for ACR.

Role of simple biomarkers in predicting fibrosis progression in HCV infection.

AIM: To examine the accuracy of the aspartate aminotransferase (AST)/Platelet Ratio Index (APRI) and FIB-4, in predicting longitudinal changes in liver histology in hepatitis C virus (HCV) patients. METHODS: Patients that underwent repeat liver biopsies at least 1 year apart from 1999 to 2007 were identified. Liver fibrosis was staged on needle core biopsies evaluated by a single expert liver pathologist. Only laboratory values within 3 mo of the liver biopsies were used. RESULTS: Thirty-six patients met the inclusion criteria with 50% stage 1 on initial biopsy, 25% stage 2, and 22% stage 3. Nineteen of 36 (53%) had progression of fibrosis on repeat biopsies, while 16 (44%) showed no change in stage, and one (3%) showed improvement. Patients that showed progression of fibrosis had significantly higher alanine aminotransferase and aspartate aminotransferase levels than the group that did not show progression. A significant correlation was seen between change in stage of fibrosis and change in APRI (r² = 0.39, P = 0.00001) and a change in FIB-4 (r² = 0.31, P = 0.00004). A change in APRI (ΔAPRI) of 0.18 had 80% positive predictive value (PPV) and 67% negative predictive value (NPV) for progression of fibrosis. A change in FIB-4 (ΔFIB-4) of 0.39 had 75% PPV and 75% NPV for predicting progression of fibrosis. CONCLUSION: ΔAPRI and ΔFIB-4 parallel changes in fibrosis progression, and could be useful tools for clinicians in following patients with active chronic HCV infection.