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Biomedical subjects

Christine M Litwin

Publications and source records attributed to Christine M Litwin.

At least 19 recordsLinked to original sources
PubMed2006

Serological diagnosis of Trypanosoma cruzi: evaluation of three enzyme immunoassays and an indirect immunofluorescent assay.

Chagas' disease is an important cause of heart failure in Latin America, but is rare in the United States. The immigration of persons from endemic countries increases the potential of encountering patients with the disease. Concerns have also been raised about the introduction of Trypanosoma cruzi, the parasite that causes the disease, into the blood supply and during organ transplantation. To compare Chagas' antibody tests that are available in the United States, we evaluated three IgG ELISAs, CeLLabs T. cruzi ELISA, Hemagen Chagas' kit and IVD Research Chagas' Serum Microwell ELISA, and MarDx indirect immunofluorescent assays. The CeLLabs and Hemagen IgG ELISAs had 100 % agreement, sensitivity and specificity. The IVD Research IgG ELISA had 94.6 % agreement, 100 % sensitivity and 93 % specificity.

Adult↗
PubMed2006

Analysis of serum antibodies in patients suspected of having inflammatory bowel disease.

Inflammatory bowel disease (IBD) is the general term used for a heterogeneous group of intestinal disorders, including Crohn's disease (CD) and ulcerative colitis (UC). Serological markers such as anti-Saccharomyces cerevisiae antibodies (ASCA) and atypical perinuclear antineutrophilic cytoplasmic antibody (atypical pANCA) have proven useful in the diagnosis and differentiation of CD and UC. Immunoglobulin A (IgA) antibody directed against the outer membrane protein C (OmpC) of Escherichia coli is said by one group to have clinical utility in diagnosing IBD, specifically in ASCA-negative CD patients. Our objective in this study was to compare the results obtained from two separate laboratories offering similar IBD tests using sera from suspected IBD patients. One hundred ninety-seven sera received for IBD testing were included in the study. The agreement between the two laboratories was 93.4% for ASCA IgA, 90.9% for ASCA IgG, and 87.8% for atypical pANCA IgG. There were 25 sera with ASCA-negative/OmpC-positive results reported by one laboratory. Thirteen of these 25 (52.0%) ASCA-negative/OmpC-positive sera were also atypical pANCA positive (9 as determined by both laboratories, 3 by one, and 1 by the other). Atypical pANCA antibody is found primarily in IBD patients with UC and colon-limited CD (Crohn's colitis). We conclude that the ASCA and atypical pANCA assays showed good agreement between the two laboratories, but the data for ASCA-negative/OmpC-positive sera suggest that many (52.0%) of these patients were more likely to have had UC or Crohn's colitis based on the presence of an atypical pANCA.

Adult↗
PubMed2006

Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis.

Recently, turbidimetric immunoassays for detecting and quantifying kappa and lambda free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound kappa and lambda light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the kappa-FLC and lambda-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal kappa/lambda FLC ratios using the turbidimetric immunoassays. In conclusion, the kappa and lambda FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the kappa/lambda ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal kappa/lambda FLC ratios.

Antibodies, Monoclonal↗
PubMed2006

Performance characteristics of six IMMULITE 2000 TORCH assays.

TORCH is an acronym for Toxoplasma gondii (Toxo), other microorganisms (eg, syphilis), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) that are associated with congenital abnormalities from maternal infection. We evaluated linearity, imprecision, and comparison with commercially available methods of the IMMULITE 2000 (Diagnostic Products, Los Angeles, CA) Toxo IgG, Toxo IgM, RV IgG, RV IgM, CMV IgG, and HSV IgG assays. Linearity and imprecision results were acceptable. The IMMULITE 2000 assays show good concordance with other commercially available methods except for Toxo IgM and RV IgM. Toxo IgM showed better concordance with a consensus of 3 of 4 (Access, Beckman Coulter, Fullerton, CA; IMMULITE 2000; Platelia, Bio-Rad Laboratories Diagnostics Group, Redmond, WA; and Vidas, bioMerieux, Hazelwood, MO) assays than with Access alone. The RV IgM assay showed better concordance with the Zeus method than with the Diamedix method (Diamedix, Miami, FL). The IMMULITE 2000 TORCH assays studied show acceptable performance and are suitable for routine clinical use. Some commercial assays for Toxo IgM and RV IgM show rather poor concordance.

Animals↗
PubMed2006

Comparison of complement fixation with two enzyme-linked immunosorbent assays for the detection of antibodies to respiratory viral antigens.

We compared complement fixation (CF) for the measurement of antibodies against influenza A, influenza B, respiratory syncytial virus (RSV), human adenovirus, and parainfluenza viruses 1, 2, and 3 (para-1, para-2, and para-3) with 2 enzyme-linked immunosorbent assays (ELISA kits, A and B). The IgG ELISA kits compared very well with each other except for the influenza A and B IgG ELISAs. The IgG ELISAs, in general, did not agree with CF In contrast, the IgM ELISAs compared well with CF and each other except for the consensus parainfluenza panel from ELISA B. The poor agreement of the IgG ELISAs with the CF test can be explained by the increased sensitivity of the ELISAs and differences between CF antigens and the ELISA antigens. The influenza A and influenza B ELISA antigens differed between both kits, which may explain their poor agreement. The ELISA is a suitable replacement for CF, providing greater sensitivity, isotype specificity, and ease of use.

Antigens, Viral↗
PubMed2006

Evaluation of an in vitro assay for interferon gamma production in response to the Mycobacterium tuberculosis-synthesized peptide antigens ESAT-6 and CFP-10 and the PPD skin test.

We compared the tuberculin skin test (TST) to QuantiFERON-TB (QFT) and QuantiFERON-TB Gold (QFT-G) for the detection of latent tuberculosis. The QFT-G uses synthesized early secretory antigenic target 6 and culture filtrate protein 10 peptide antigens instead of purified protein derivative (PPD) antigens. The study included 137 adults in 3 groups: 1 (n = 81), at low risk for Mycobacterium tuberculosis (TB) and not vaccinated for Mycobacterium bovis bacillus Calmette-Guérin (BCG); 2 (n = 30), probably had TB exposure and were BCG vaccinated; and 3 (n = 26), at low risk for TB, not BCG vaccinated, but previously had a positive TST result. Positive results were as follows: group 1: TST 3 (3.7%); QFT 9 (11.1%); and QFT-G, 0 (0.0%); group 2: TST 26 (86.7%); QFT, 15 (50.0%); and QFT-G, 5 (16. 7%); and group 3: TST, 26 (100.0%); QFT, 13 (50.0%); and QFT-G, 9 (34.6%). The QFT-G demonstrated less cross-reactivity with BCG antigen and was more specific than QFT and TST in low-risk individuals.

Adult↗
PubMed2005

Evaluation of a western blot method for the detection of Yersinia antibodies: evidence of serological cross-reactivity between Yersinia outer membrane proteins and Borrelia burgdorferi.

Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.

Antibodies, Bacterial↗
PubMed2005

Identification, cloning, and expression of the CAMP-like factor autotransporter gene (cfa) of Bartonella henselae.

The CAMP reaction was first described by Christie et al. (R. Christie, N. E. Atkins, and E. Munch-Petersen, Aust. J. Exp. Biol. 22:197-200, 1944) as the synergistic lysis of sheep red blood cells by Staphylococcus aureus sphingomyelinase and CAMP factor (cohemolysin), a secreted protein from group B streptococci. We observed a CAMP-like reaction when Bartonella henselae was grown in close proximity to S. aureus on 5% sheep blood agar. This study describes the cloning, sequencing, and characterization of a CAMP-like factor autotransporter gene (cfa) from B. henselae. A cosmid library of B. henselae ATCC 49793 was constructed using SuperCos1 in Escherichia coli XL1-Blue MR. Cosmids were screened for the CAMP reaction, and a quantitative cohemolysis microtiter assay was developed using purified sphingomyelinase. Cosmid clones with the strongest cohemolytic reaction had similar restriction enzyme patterns. A DNA fragment that expressed the cohemolysin determinant was subcloned in a 7,200-bp StuI-BamHI fragment which contained a 6,024-bp open reading frame. The deduced amino acid sequence showed homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane. They contain an N-terminal passenger region, the alpha-domain, and a C-terminal transporter region, the beta-domain. The alpha-domain contained four, nearly identical 42-amino-acid repeats and showed homology to the family of RTX (repeat in toxin) hemolysins. The concentrated supernatant of the recombinant strain expressed a protein with a molecular mass of 180 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis consistent with the calculated molecular weight of the secreted alpha-domain. In conclusion, we have characterized a novel secreted cohemolysin autotransporter protein of B. henselae.

Amino Acid Sequence↗
PubMed2004

The Bartonella henselae sucB gene encodes a dihydrolipoamide succinyltransferase protein reactive with sera from patients with cat-scratch disease.

Bartonella henselae is a recently recognized pathogenic bacterium associated with cat-scratch disease, bacillary angiomatosis and bacillary peliosis. A recombinant clone expressing an immunoreactive antigen of B. henselae was isolated by screening a genomic DNA cosmid library by Western blotting with sera pooled from patients positive for B. henselae IgG antibodies by indirect immunofluorescence (IFA). The deduced amino acid sequence of the 43.7 kDa encoded protein was found to be 76.3 % identical to the dihydrolipoamide succinyltransferase enzyme (SucB) of Brucella melitensis. SucB has been shown to be an immunogenic protein during infections by Brucella melitensis, Coxiella burnetii and Bartonella vinsonii. The agreement between reactivity with a recombinant SucB fusion protein on immunoblot analysis and the results obtained by IFA was 55 % for IFA-positive sera and 88 % for IFA-negative sera. Cross-reactivity was observed with sera from patients with antibodies against Brucella melitensis, Mycoplasma pneumoniae, Francisella tularensis, Coxiella burnetii and Rickettsia typhi.

Acyltransferases↗
PubMed2004

Heterophile antibody interference in a multiplexed fluorescent microsphere immunoassay for quantitation of cytokines in human serum.

While modern immunoassays provide sensitive and specific means for the quantitation of cytokines in biological fluids, heterophile antibodies are still a well-recognized cause of interference in the measurement of cytokines in these assays. We have developed a multiplexed fluorescent microsphere immunoassay for the simultaneous quantification of 10 cytokines in only 75 microl of serum. During the development of this multiplexed assay, the amount of assay interference due to heterophile antibodies was also determined, and methods for detecting heterophile interference and minimizing its effect were incorporated into the assay. Heterophile antibodies resulted in significantly elevated cytokine values compared to those of normal blood bank samples. These falsely elevated values, and thus the components of the assay the heterophile antibodies were binding to, were identified through the use of internal controls. This information was then used to design assay-specific blockers and absorbents that were shown to significantly reduce falsely elevated cytokine values while not affecting the standard and control values. The fluorescent multiplexed microsphere-based immunoassay can be used to quantitate multiple cytokines from a single sample and should be a useful tool in furthering our understanding of the role of cytokines in disease processes.

Antibodies, Heterophile↗
PubMed2004

Evaluation of multiplexed fluorescent microsphere immunoassay for detection of autoantibodies to nuclear antigens.

Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

Antibodies, Antinuclear↗
PubMed2004

Evaluation of an in vitro assay for gamma interferon production in response to Mycobacterium tuberculosis infections.

The tuberculin skin test (TST) is the "gold standard" for detecting infection with Mycobacterium tuberculosis. We compared the TST using purified protein derivative to the QuantiFERON-TB test (QFT). Two groups were examined. Group 1 individuals (n = 66) (low risk) were at low risk for exposure to M. tuberculosis and were not Mycobacterium bovis BCG vaccinated. Group 2 (n = 29) include individuals who were likely to have been exposed to a high prevalence of M. tuberculosis infections and were BCG vaccinated. Group 1 individuals were given a TST. Group 2 individuals were not given a TST because of possible adverse reactions. A 10- to 15-mm indurated area 48 h after TST was considered positive. A positive QFT result was defined as a significant gamma interferon response to M. tuberculosis antigen, Mycobacterium avium antigen, and a nonspecific mitogen stimulus and no response in the negative control. In group 1, 60 of 66 individuals (90.9%) were negative by both methods, and 1 person was positive by both methods. There was one QFT-negative, TST-positive case, one QFT-positive, TST-negative case, and three conditional QFT-positive, TST-negative cases. In group 2, 12 of 29 (41.4%) were positive by QFT and considered likely to be TST positive because of prior BCG vaccination. QFT testing in our low-risk group resulted in an agreement of 96.8%, a sensitivity of 50%, and a specificity of 98.4% compared with TST results. QFT testing with TST in low-risk groups can aid in the detection of latent M. tuberculosis infections.

Adult↗
PubMed2004

Evaluations of commercial West Nile virus immunoglobulin G (IgG) and IgM enzyme immunoassays show the value of continuous validation.

West Nile virus was introduced into the United States in 1999 and in only four seasons has become endemic east of the Rocky Mountains. Recently, immunoglobulin M (IgM)-capture enzyme immunoassays for the detection of West Nile virus-specific IgM and indirect IgG enzyme immunoassays for the detection of IgG antibodies against West Nile virus were made available from Focus Technologies and PANBIO, Inc. We evaluated these commercial IgG and IgM test systems and determined agreement, sensitivity, and specificity for the assays, compared to immunofluorescence assay and the Centers for Disease Control and Prevention's IgM-capture enzyme-linked immunosorbent assay (ELISA). Initially, the Focus and PANBIO IgM enzyme immunoassays had at least 95% agreement, sensitivity, and specificity, and, based on the 95% confidence intervals, both IgM-capture assays performed similarly. The IgG assays also performed well, although the Focus IgG assay demonstrated greater specificity (98.8%) and the PANBIO IgG assay demonstrated greater sensitivity (99.3%). However, for 400 samples consecutively submitted for West Nile virus antibody testing during 2 days of the 2003 West Nile virus season, agreement, clinical sensitivity, and clinical specificity were 93.1, 98.0, and 92.4%, respectively, for the PANBIO IgM assay and were 97.4, 100.0, and 97.1%, respectively, for the Focus IgM assay. The specificities observed in this second evaluation equates to an overall false-positivity rate of 6.3% in the PANBIO West Nile virus IgM-capture ELISA versus 2.5% with the Focus West Nile virus IgM-capture ELISA. This experience demonstrates the importance of continuously evaluating the performance of an assay in order to detect any changes in assay performance as the test population evolves.

Adult↗
PubMed2003

Comparison of two commercial enzyme-linked immunosorbent assays with an immunofluorescence assay for detection of Legionella pneumophila types 1 to 6.

Members of the genus Legionella are characterized as gram-negative, motile, freshwater-dwelling bacteria that were responsible for a pneumonia outbreak among American Legion members in 1976. Because clinicians routinely order serologic testing for Legionella pneumophila serogroups 1 to 6 as a screen for possible L. pneumophila infections, we evaluated the Wampole Laboratories L. pneumophila type 1 to 6 immunoglobulin G (IgG) and IgM combined enzyme-linked immunosorbent assay (ELISA) and the Zeus Scientific L. pneumophila type 1 to 6 IgG-IgM-IgA multispecific combined ELISA systems and compared them to an IgG-specific immunofluorescence assay (IFA) for L. pneumophila serogroups 1 to 6. The Centers for Disease Control and Prevention recommends that the positive titer cutoff for an IFA be 1:256. Regardless of where the positive IFA cutoff titer is placed, however, the sensitivity of both commercial assays was below what would be acceptable for a screening assay. With a 1:256 IFA titer as the positive cutoff, the agreement, sensitivity, and specificity of the Wampole ELISA were 74.6, 21.4, and 98.4%, respectively. The agreement, sensitivity, and specificity of the Zeus ELISA were 72.6, 10.5, and 100.0%, respectively. We recommend that any laboratories attempting to replace an IFA type 1 to 6 screen with an alternative ELISA carefully investigate the sensitivity of the replacement assay.

Antibodies, Bacterial↗
PubMed2003

Detection of IgG and IgM to West Nile virus. Development of an immunofluorescence assay.

West Nile virus (WNV) had its first recorded appearance in the western hemisphere in 1999 and has continued to spread across the United States, necessitating the development of serologic procedures to diagnose infection. We developed an immunofluorescence assay (IFA) protocol for the detection of WNV-specific IgG and IgM antibodies in serum and cerebrospinal fluid (CSF) specimens. We tested 82 serum and 16 CSF samples and compared the results with WNV IgG enzyme-linked immunosorbent assay (ELISA) and IgM antibody-capture (MAC) ELISA results. Agreement, clinical sensitivity, and clinical specificity for the IgG IFA were 92%, 100%, and 90%, respectively, and 98%, 96%, and 100% for the IgM IFA, respectively. Extensive arbovirus cross-reactions occurred in the IgG assays, but only minimal cross-reactions were observed in the IgM assays. The IFA protocol described herein is a cost-effective and sensitive alternative to ELISA and MAC-ELISA for the serologic diagnosis of WNV infection.

Antibodies, Viral↗
PubMed2002

Comparison of four enzyme immunoassays for the detection of immunoglobulin G antibody against glomerular basement membrane.

Goodpasture syndrome is a life-threatening autoimmune kidney and pulmonary disease that is characterized by pulmonary hemorrhage, renal failure, and the presence of autoantibodies against glomerular basement membrane (GBM) by indirect fluorescent antibody (IFA) techniques. In 1988, these antibodies were found to be specific for the noncollagen region of the alpha3 collagen IV chain. The antigen is characterized by a restricted tissue distribution occurring mainly in the kidneys and lungs. Specific enzyme immunoassays (EIAs) for anti-GBM have now been developed for in vitro diagnostic use in the laboratory. Our objective in this study was to compare the results obtained using four different EIAs for detecting anti-GBM IgG antibody with those using the standard IFA method using tissue from human kidney. Thirty-two patients with suspected Goodpasture syndrome, and 10 control sera were included in the study. GBM EIAs were purchased from or donated by the following vendors: Scimedx Corporation (Denville, NJ), INOVA Diagnostics (San Diego, CA), The Binding Site (Birmingham, England), and Wieslab (distributed by DiaSorin, Inc., Stillwater, MN). Percent agreement, sensitivity, and specificity were calculated for each EIA as compared to IFA. The results were as follows: Binding Site: 83.3, 100.0, and 72.0%; Wieslab: 95.2, 94.1, and 96.0%; INOVA: 96.2, 100.0, and 92.3%; and Scimedx: 81.0, 100.0, and 68.0%. We conclude that the INOVA and Wieslab GBM IgG EIAs compared well with the standard GBM IFA method using tissue from human kidney. The Scimedx and Binding Site EIAs had eight and seven false-positive results, respectively, when compared to GBM IFA using human kidney. The authors recommend conducting thorough evaluations of EIAs that screen for anti-GBM antibody before their implementation in the clinical laboratory. We also recommend confirming GBM EIA-positive sera by the standard IFA method using tissue from human kidney.

Anti-Glomerular Basement Membrane Disease↗
PubMed2002

A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides.

We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F; 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.

Antibodies, Bacterial↗