PubMed Health⌕ Search

Biomedical subjects

C M Litwin

Publications and source records attributed to C M Litwin.

At least 19 recordsLinked to original sources
PubMed2001

Comparison of four enzyme immunoassays with a western blot assay for the determination of type-specific antibodies to herpes simplex virus.

Most current enzyme immunoassays (EIAs) differentiate inadequately between types 1 and 2 herpes simplex virus (HSV) antibodies since significant cross-reactivity exists. We compared 4 IgG type-specific EIAs using a Western blot assay for resolution of discrepant results. The Diamedix had sensitivities of 100% for types 1 and 2 but specificities of only 71% and 61%, respectively. The cross-reactivity rate was 82% in positive samples tested. For HSV types 1 and 2, the Zeus sensitivities were 92% and 98%, respectively; specificities were 72% and 79%, respectively; the cross-reactivity rate was 54%. For HSV types 1 and 2, the Wampole sensitivities were 98% and 95%, respectively; specificities were 68% and 85%, respectively; the cross-reactivity rate was 47%. For HSV types 1 and 2, the Meridian sensitivities were 98% and 90%, respectively; specificities were 96% and 100%, respectively; no cross-reactivity was found between positive samples tested. While the Diamedix, Zeus, and Wampole assays showed good sensitivity, they lacked type specificity. The Meridian EIA offers the highest specificity along with no observed cross-reactivity. This EIA may be an easier, reliable alternative to Western blot for the determination of HSV type-specific antibodies.

Antibodies, Viral↗
PubMed2000

Cloning and characterization of vuuA, a gene encoding the Vibrio vulnificus ferric vulnibactin receptor.

The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and in V. vulnificus, transcriptional regulation by iron depends on the fur gene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V. vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae vibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V. vulnificus. Analysis of the DNA sequence of the vuuA promoter region demonstrated a sequence identical to the upstream Fur box of V. cholerae viuA. Northern blot analysis showed that the transcript was strongly regulated by iron. The amino acid sequence of VuuA was 74% identical to the sequence of V. cholerae ViuA and was homologous to those of several TonB-dependent outer membrane receptors. An internal deletion of the V. vulnificus vuuA gene resulted in the loss of expression of the 72-kDa protein and the loss of the ability to use transferrin or vulnibactin as a source of iron. This mutant showed reduced virulence in an infant mouse model. Introduction of a plasmid containing the complete viuA coding sequence and 342 bp of upstream DNA into the mutant restored ferric vulnibactin and ferric transferrin utilization to the mutant.

Amides↗
PubMed2000

Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control.

Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.

Amniotic Fluid↗
PubMed1999

Comparison of capillary zone and immunosubtraction with agarose gel and immunofixation electrophoresis for detecting and identifying monoclonal gammopathies.

Capillary zone electrophoresis (CZE) and immuno-subtraction electrophoresis (ISE) were evaluated for ability to detect and immunotype monoclonal proteins, compared with agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE), respectively. Six hundred seventeen serum samples were analyzed with CZE and AGE to determine sensitivity and specificity in detecting IFE-confirmed monoclonal gammopathies. Both techniques detected all monoclonal spikes due to IgM (n = 8), IgG (n = 38), and free light chains (n = 3). Agarose gel electrophoresis, however, detected only 11 of 14 (79%) IgA monoclonal spikes detected with CZE. In a second study, 78 serum samples, 48 of which had a monoclonal gammopathy confirmed with IFE, were evaluated with ISE. Only 60% to 75% of the monoclonal gammopathies were correctly immunotyped with ISE by 4 readers blinded to the IFE immunotype. Thus CZE was more sensitive than AGE in detecting low concentrations of monoclonal proteins, but ISE is less accurate than IFE in determining the immunotype of the monoclonal gammopathy.

Autoanalysis↗
PubMed1999

Comparison of three different methods for measuring classical pathway complement activity.

The complement system plays an important role in host defense against infection and in most inflammatory processes. The standard 50% hemolytic complement (CH50) assay is the most commonly used method of screening patient sera for functional activity of the classical complement pathway. Our objective in this study was to compare two newer methods (the enzyme immunoassay and the liposome immunoassay) to a commercial CH50 assay for measuring total classical complement activity. We conclude that both newer methods compare well with a CH50 assay and are equally sensitive in screening routine clinical sera.

Adult↗
PubMed1999

Heterophile antibodies to bovine and caprine proteins causing false-positive human immunodeficiency virus type 1 and other enzyme-linked immunosorbent assay results.

Heterophile antibodies are a well-recognized cause of erroneous results in immunoassays. We describe here a 22-month-old child with heterophile antibodies reactive with bovine serum albumin and caprine proteins causing false-positive results to human immunodeficiency virus type 1 and other infectious serology testing.

Animals↗
PubMed1999

Gender differences in postinfarction left ventricular remodeling.

OBJECTIVE: Previous studies suggest that gender affects the adaptive responses of the heart to some forms of cardiac overload. It is unknown whether gender influences left ventricular (LV) remodeling after myocardial infarction (MI). METHODS: We performed transthoracic echocardiographic-Doppler examinations in age-matched male (n = 17) and female (n = 16) rats before, and 1 and 6 weeks after transmural MI or sham surgery. RESULTS: Following large MI (male = 45 +/- 1% LV circumference vs. female = 48 +/- 4%, p = NS), both male and female rats developed progressive LV dilatation. Infarctions caused a similar degree of global and regional LV systolic dysfunction in males and females. Male rats had significant increases in the thickness of the noninfarcted posterior wall by 6 weeks after MI. However, posterior wall thickness did not change in the infarcted female rats. Average myocyte diameter in the noninfarcted region of the heart was also greater in male than female MI rats. The combination of increased cavity size with little change in wall thickness resulted in a greater decline in relative wall thickness in the female rats compared to the males. Male rats with MI showed progressively restricted LV diastolic filling as assessed by transmitral Doppler recordings. Female rats had less of an increase in the ratio of early to late transmitral velocities and less of an increase in the E wave deceleration rate after MI. CONCLUSIONS: Female rats showed a different pattern of LV remodeling than males with less of an increase in thickness of the noninfarcted portions of the left ventricle than males, but comparable LV cavity enlargement and systolic dysfunction. Despite similar infarct size, females developed less pronounced abnormalities of LV diastolic filling. We hypothesize that the gender-related differences in postinfarction LV remodeling may contribute to the different LV filling patterns, and might ultimately relate to differences in clinical outcome.

Animals↗
PubMed1998

Comparison of an inhalant allergy immunoassay with allergen specific IgE determinations in human serum.

The diagnosis of inhalant allergy can be elusive, with symptoms resembling viral or bacterial infection, as well as immunologic deficiency. In this study an inhalant allergy immunoassay was investigated as a possible screen to rule in or out respiratory inhalant allergy in patients with allergic-type symptoms. The results of this screen were compared in 192 serum specimens submitted to our laboratory for specific IgE allergy testing and 73 blood bank samples. When the discrepant results of the inhalant allergy immunoassay were resolved by Western blot, a final sensitivity of 94.7% and specificity of 97.5% was calculated. We have found this inhalant allergy immunoassay to be an effective screen for detecting inhalant allergies, and believe it to be a useful tool for the primary care physician or non-allergist trying to differentiate inhalant allergens from chronic sinusitis or other causes of sinopulmonary congestion.

Allergens↗
PubMed1998

Cloning and characterization of an outer membrane protein of Vibrio vulnificus required for heme utilization: regulation of expression and determination of the gene sequence.

Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis.

Amino Acid Sequence↗
PubMed1997

Detection of Epstein-Barr viral DNA in serum using rapid-cycle PCR.

Our study describes the comparison of a rapid nested PCR assay to standard serology techniques for the detection of Epstein-Barr virus (EBV) in serum. The sera of 81 patients with suspected EBV infection were analyzed; 54 were positive for one or more of the standard serology markers, i.e., IgM viral capsid antigen (VCA), IgG-VCA, Epstein-Barr nuclear antigen 1 (EBNA-1), and early antigen (EA), and 27 were negative for all serology markers. The sera from 15 normal healthy blood donors were also included. No EBV DNA was detected in any of the 15 blood donor samples or in any of the 27 samples with negative serology results. Eleven samples (20%) of the 54 with positive EBV serology results were positive for EBV DNA. Of these samples, 9 were EBV IgM-VCA positive and anti-EBNA negative, suggesting acute infection. One of the 11 samples had high titers of IgM-VCA, IgG-VCA, anti-EBNA, and anti-EA. The last of the 11 samples was from a patient with acute infectious mononucleosis without sufficient sample volume for EBV serology testing. Seventeen of the total 96 samples from the study were IgM-VCA positive and anti-EBNA negative and 9 of these 17 samples (53%) tested positive for EBV DNA. These data suggest that the detection of EBV DNA by PCR in serum may be a useful indicator of active infection rather than latent virus.

Antibodies, Viral↗
PubMed1997

Rapid polymerase chain reaction-based detection of the causative agent of cat scratch disease (Bartonella henselae) in formalin-fixed, paraffin-embedded samples.

Bartonella (formerly Rochalimaea) henselae (Bh) plays a central role in cat scratch disease. A polymerase chain reaction (PCR)-based assay that can detect Bh DNA in formalin-fixed, paraffin-embedded (FF-PE) samples would have utility in the evaluation of processed lymph nodes suggestive of this disorder. Fresh or FF-PE cultures of Bh and related species were analyzed. Thirteen lymph nodes (12 FF-PE and one fresh cell suspension) with necrotizing suppurative granulomatous inflammation and seven FF-PE negative control lymph nodes were also evaluated. PCR was performed using a novel, hemi-nested protocol. Amplified products were analyzed by gel electrophoresis. The fresh and FF-PE Bh cultures showed a specific PCR product with an analytical sensitivity of 0.5 pg bacterial DNA. Seven (54%) of 13 clinical lymph node samples with morphological features suggestive of cat scratch disease also had detectable Bh DNA, whereas none of the seven negative control lymph nodes yielded positive results. We have designed a rapid and sensitive PCR test that can reliably detect Bh DNA in fresh and FF-PE samples. Our findings indicate that this assay has clinical utility in the diagnosis of cat scratch disease.

Adult↗
PubMed1997

Characterization of endemic strains of Shigella sonnei by use of plasmid DNA analysis and pulsed-field gel electrophoresis to detect patterns of transmission.

Shigellosis is hyperendemic in Utah. Most isolates are Shigella sonnei, making it difficult to identify epidemiologic clustering. To better define transmission, molecular markers and epidemiologic data were examined for 90 cases. Plasmid analysis and pulsed-field gel electrophoresis (PFGE) of the S. sonnei isolates identified 11 and 4 patterns, respectively. Plasmid pattern I infections occurred in 8 day care centers over a 6-month period, suggesting spread between centers. Plasmid pattern III was isolated from children at 3 additional centers and pattern IV was associated with another day care center, suggesting different outbreaks. By PFGE, plasmid groups I and XI appeared identical, as were plasmid groups II and V; plasmid group X had a unique pattern. Plasmid groups III, IV, and VII-IX were closely related PFGE subtypes. Both plasmid analysis and PFGE allow better characterization of S. sonnei transmission patterns of "endemic" strains and could lead to improved control measures.

DNA, Bacterial↗
PubMed1997

Immunologic response to Bartonella henselae as determined by enzyme immunoassay and Western blot analysis.

Bartonella henselae is now regarded as the etiologic agent of cat-scratch disease and a cause of bacillary angiomatosis. We examined the human immune response to Bartonella henselae infection using a newly developed enzyme immunoassay (EIA) and a Western blot procedure using outer-membrane proteins. The EIA showed 98.6% and 91.4% agreement with an indirect fluorescence method (IFA) for detection of IgM and IgG antibodies, respectively. By using Western blot analysis, reactivity to an 8-kd band showed significant correlation with positive results by the IgM IFA and EIA. In contrast, reactivity to 209-, 208.5-, 208-, 116-, and 80-kd bands was identified only in positive IgG IFA serum samples. The EIA and Western blot should be useful tests in determining the antibody response to B. henselae infection and may also be important in determining the critical epitopes in the host-parasite interaction of this organism.

Adolescent↗
PubMed1997

Immunoglobulin A antibodies to Helicobacter pylori.

Serological testing for immunoglobulin G (IgG) antibodies to Helicobacter pylori has proven useful in supporting the diagnosis of infection with this organism, but the clinical value of IgA antibodies in H. pylori-related gastritis remains controversial. The purpose of our study was to determine the frequency of IgA-positive IgG-negative patients with symptoms of gastrointestinal (GI) disorders, thus assessing the clinical utility of IgA testing for H. pylori-related gastritis. It was found previously that the frequency of infected individuals in this category (IgA positive and IgG negative) is about 2%, but a large number of IgG-negative patients with GI disorders suggestive of H. pylori infection have not been investigated until now.

Antibodies, Bacterial↗
PubMed1996

Anti-glomerular basement membrane disease: role of enzyme-linked immunosorbent assays in diagnosis.

We compared two enzyme-linked immunosorbent assay (ELISA) tests with indirect immunofluorescence (IFA) for the measurement of anti-glomerular basement membrane (GBM) antibodies to study their sensitivity and specificity. The first ELISA test kit compared well to IFA (96.4% agreement, 93.3% sensitivity, 97.0% specificity). The second ELISA kit was much less sensitive (87.8% agreement, 63.3% sensitivity, 92.2% specificity). ELISA tests for the measurement of anti-GBM antibodies allow for quantitation and may have greater sensitivity than IFA. Sensitivity, however, may depend on the commercial test used.

Adolescent↗
PubMed1996

Screening for antinuclear antibodies by enzyme immunoassay.

Indirect fluorescent antibody (IFA) ia most widely used method in clin clinical laboratories to screen for autoantibodies against a wide variety of nuclear antigens. Recently, a number of antinuclear antibody (ANA) enzyme immunoassay (EIA) screens have become commercially available and claim to be an alternative method to screen for ANAs. Given the subjectivity of technical interpretation of IFA and the high number of ANA negative samples, a suitable EIA method for ANA screening would be beneficial to clinical laboratories with large samples volumes. Five ANA EIA screens were compared (Elias, Helix, Sanofi, TheraTest and Zeus) to IFA using a human epithelial cell line (HEp-2). Sera from 601 patients submitted to our reference laboratory for autoimmune testing, and from 202 normal healthy blood donors, were included in this study. Samples with discordant results between IFA and EIA were further analyzed using single antigen EIAs for SSA, SSB, Sm, RNP, Scl-70, histones, dsDNA, and ssDNA. Analyses were based on clinically significant IFA titers of > or equal to 1:160 as positive and <1:40 as negative. When compared to IFA, agreement, sensitivity and specificity for each ANA EIA screen were as follows: Elias: 87.0%, 69.5% and 97.9%; Helix: 94.6%, 90.2%, and 97.3%; Sanofi: 95.0%, 93.7%, and 95.9%; TheraTest: 95.3%, 97.7%, and 93.5%; Zeus: 87.1%, 96.2%, and 81.4%, respectively. In conclusion, screening for ANAs by EIA using several commercial assays was both sensitive and specific when compared to IFA. Moreover, the EIA is objective and much less labor intensive when screening a large number of clinical specimens. None of the EIAs were 100% sensitive and, thus, may fail to detect a few of the nonspecific ANAs that demonstrate atypical as well as classical IFA patterns. The advantages of employing these nonsubjective assays to screen out the vast majority of ANA negative sera is clear. The authors still recommend confirming titers and patterns of sera with positive EIA screens using classical IFA methods employing HEp-2 cells.

Antibodies, Antinuclear↗
PubMed1996

Polymerase chain reaction detection of Lyme disease: correlation with clinical manifestations and serologic responses.

The authors have developed a simple, nested polymerase chain reaction (PCR) assay for amplification of an outer surface protein A (OspA) gene fragment of Borrelia burgdorferi using rapid temperature cycling and ethidium bromide detection on agarose gels, and applied it to the diagnosis of Lyme disease in humans. With denaturing and annealing temperature spikes instead of holds, cycle times were less than 20 minutes for a 30-cycle amplification. Using this rapid cycle PCR technique, as few as 5 spirochetes per mL of phosphate buffered saline were detected. In addition, B burgdorferi DNA was detected from spirochetes that had been spiked into one of several types of human body fluids including serum, synovial fluid, and cerebrospinal fluid (CSF). A number of clinical samples, which had been tested for Lyme immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody were also examined. In 29 serologic positive samples (14 IgG and IgM positive, 9 IgM alone and 6 IgG alone), B burgdorferi DNA was not detected. In contrast, nine serum samples and one synovial fluid from patients with definite clinical features of Lyme disease were found to be negative by EIA and Western blot analysis for IgG and IgM antibody, but contained B burgdorferi DNA, as detected by PCR. Polymerase chain reaction analysis of serum and synovial fluid may be of significant diagnostic value in Lyme disease, especially in the absence of a serologic response in early, partially treated and seronegative chronic disease. This is the first study to report an association between PCR positivity and the absence of a serologic response to Lyme borreliosis.

Base Sequence↗