Use of "<Emphasis Type="Italic">biokit</Emphasis>HSV-2 Rapid Assay" to improve the positive predictive value of Focus HerpeSelect HSV-2 ELISA

Background  

Commercially available assays to detect antibodies to the herpes simplex virus type 2 (HSV-2)-specific glycoprotein gG-2 have markedly improved serologic diagnosis of HSV-2 infection. However, even tests with high specificity can have low positive predictive values in low prevalence populations. HSV-2 is a chronic, life-long viral infection that requires both medical attention and potential alterations in health care strategy. As such, the concern for false positive diagnoses is high confirmatory testing is routine for other viral serologies such as HIV and hepatitis C. We evaluated such a strategy for HSV-2 serology by using an easily performed commercial test, biokitHSV-2 rapid test ("Biokit"; Biokit USA, Lexington MA) as a confirmatory test for the widely used gG-2 specific serology ("Focus;" HerpeSelect HSV-2 ELISA; Focus Diagnostics, Cypress CA).     

Comparison of two interferon gamma release assays in the diagnosis of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> infection and disease in The Gambia

Background  

IFN-γ Release Assays (IGRAs) have been licensed for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia.     

Sensitivity and specificity of monoclonal and polyclonal immunohistochemical staining for West Nile virus in various organs from American crows (<Emphasis Type="Italic">Corvus brachyrhynchos</Emphasis>)

Background  

Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection.     

Higher incidence of persistent chronic infection of <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> among coronary artery disease patients in India is a cause of concern

Background  

There is growing evidence that Chlamydia pneumoniae may be involved in the pathogenesis of atherosclerosis, as several studies have demonstrated the presence of the organism in atherosclerotic lesions. C. pneumoniae infections, which are especially persistent infections, have been difficult to diagnose either by serological methods or isolation of the organism from the tissue. Nucleic Acid Amplification tests (NAATs) has emerged as an important method for detecting C. pneumoniae. Inspite of high prevalence of C. pneumoniae specific antibodies in coronary heart disease patients, direct detection of C. pneumoniae in circulating blood of coronary artery disease (CAD) patients by sensitive nucleic acid amplification tests nested PCR (nPCR), multiplex PCR (mPCR) has not been carried out is required. Further correlation of the presence of C. pneumoniae in blood of CAD patients with C. pneumoniae specific IgA and IgG antibodies, which may indicative of the status of infection with the progression of atherosclerosis. This will help in order to prepare strategies for the antibiotic intervention to avoid the progression towards CAD.     

Clinical significance of antinuclear antibodies. Comparison of detection with immunofluorescence and enzyme-linked immunosorbent assays

Objective. To compare the measurement of antinuclear antibodies (ANA) by immunofluorescence and by 6 different commercial enzyme-linked immunosorbent assays (ELISAs) in clearly defined patient groups and serum samples. Methods. Serum samples were derived from 3 sources: 1) patients with a known clinical diagnosis of systemic lupus erythematosus (SLE) (group 1), 2) sera with known monospecific ANA reactivity (group 2), and 3) sera from consecutive patients for whom ANA testing had been ordered (group 3). Each of these sera was tested for the presence of ANA by immunofluorescence on HEp-2 cells, and by using each of 6 commercially available ELISA ANA kits. Results. In patients with known clinical SLE, 88% were ANA positive by immunofluorescence. Positivity with the different ELISAs ranged from 62% to 90%. While most ELISAs successfully detected antibodies to SS-A, RNP, and Sm, there were significant differences between assays in the detection of anticentromere antibodies and anti-DNA. Measurement of ANA in consecutive patients for whom an ANA test was requested showed that, generally, those assays with high sensitivity for detection of SLE had a high false-positive rate, whereas those assays with a low false-positive rate failed to identify some patients with a clinical diagnosis of SLE. Conclusion. There are significant differences in the detection of ANA by immunofluorescence and different ELISA kits. Agreement between different assays is generally marginal. When ordering and interpreting an ANA test, the clinician must be familiar with the specific assay being used to measure ANA and the differences between the various ANA assays.     

Association of α‐actinin–binding anti–double‐stranded DNA antibodies with lupus nephritis

Objective

Anti–double‐stranded DNA (anti‐dsDNA) antibodies may contribute to the pathogenesis of glomerulonephritis (GN) by cross‐reacting with α‐actinin in murine models and in some patients with systemic lupus erythematosus (SLE). We therefore sought to determine possible disease associations with serologic and clinical features and to characterize this new autoantibody specificity.

Methods

One hundred patients with SLE were recruited into this multicenter study, as well as 100 rheumatic disease controls and 2,100 healthy blood donors. Clinical disease was evaluated by the SLE Disease Activity Index (SLEDAI; excluding the anti‐DNA component). Anti‐dsDNA antibodies were detected by conventional enzyme‐linked immunosorbent assay (ELISA) and by a commercial enzyme immunoassay (EIA). Anti–α‐actinin antibodies were detected by ELISA, and their specificity was confirmed by Western blotting and by indirect immunofluorescence using rat kidney sections and mesangial cells as substrates. Highly positive sera were selected for absorption experiments and were affinity‐purified for cross‐reactivity studies and measurement of antibody avidity.

Results

Sera from 62 of the SLE patients had anti‐dsDNA antibodies; 21 of these sera also had anti–α‐actinin antibodies, as compared with 1 of the 38 sera without anti‐dsDNA antibodies. Of the 22 patients with anti–α‐actinin antibodies, 10 had GN, as compared with 14 of the 78 without anti–α‐actinin antibodies (P < 0.01). In patients with GN, anti–α‐actinin, but not anti‐dsDNA, antibodies correlated with the SLEDAI score (minus the anti‐DNA component) and with treatment. The fraction of serum anti‐dsDNA antibodies that cross‐reacted with α‐actinin exhibited high avidity for dsDNA, as determined using a commercial EIA for high‐avidity anti‐dsDNA antibodies and an in‐house conventional ELISA.

Conclusion

The α‐actinin–binding antibodies are significantly associated with GN in SLE. Whether such autoantibodies may anticipate the development of this complication of SLE remains to be verified.

     

A comparative evaluation of a new fully automated assay for von Willebrand factor collagen binding activity to an established method

Introduction

Laboratory diagnosis of von Willebrand disease (VWD) is made by the measurement of von Willebrand factor (VWF) protein level and its activities. Current VWF activity tests include ristocetin cofactor and collagen binding (VWF:CB) assays.

Aim

We have undertaken an evaluation of a new fully automated VWF:CB assay relative to an established enzyme‐linked immunosorbent assay (ELISA) method.

Methods

The two analytical systems operate with different detection principles: a chemiluminescent method performed on ACL AcuStar Analyzer (the former) and a colorimetric ELISA by Asserachrom Stago (the latter) (type III collagen from human placenta). The HemosIL AcuStar VWF:CB assay is a chemiluminescent 2‐step immunoassay that uses magnetic particles coated with a type III collagen triple‐helical peptide. VWF:CB levels were determined in 50 healthy subjects and 100 VWD patients (22 type 1, 73 type 2 and 5 type 3).

Results

Eleven VWD samples reported VWF:CB values below the lower detection limit of one or both methods. The new method showed a good correlation with the ELISA method (r > .9, mean bias 3.85 IU/dL) in both healthy and VWD samples. One of 150 samples gave inconsistent results using the two assays, leading to an uncertain diagnosis of VWD type 1 (ELISA method) or type 2 MCB (fully automated method).

Conclusion

The new assay is rapid and simple to use, with its ready‐to‐use reagent cartridges. This VWF:CB assay, in addition to the measurement of VWF:Ag and VWF:RCo made on the same platform, gives additional information for the diagnosis of VWD in both nonspecialized and reference laboratories.     

Role of interferon-gamma release assays in the diagnosis of pulmonary tuberculosis in patients with advanced HIV infection

Background  

T-cell interferon-gamma release assays (IGRAs) may have a role in the diagnosis of active tuberculosis when evaluating patients for whom standard microbiology has limited sensitivity. Our objective was to examine the accuracy of a commercial IGRA for diagnosis of active tuberculosis in HIV-infected persons.     

Detection of anti-IgA antibodies using the particle gel immunoassay: a rapid test for increased patient safety

Background

Patient safety is a major issue in transfusion medicine and commands continuous efforts to develop valid control methods aiming to avoid serious transfusion-related complications. Anti-IgA antibodies can cause anaphylactic transfusion reactions in IgA-deficient individuals. Since standard quantitative methods for anti-IgA measurement require considerable time to be performed, in an emergency situation it can be a challenge to prevent or to quickly interpret and manage acute transfusion reactions suspected to be a consequence of anti-IgA. The purpose of this study was to test and validate at our transfusion centre a rapid assay for the identification of patients with anti-IgA antibodies.

Materials and methods

Forty-six samples (6 from healthy controls and 40 from IgA-deficient patients) were collected. Sera were analysed blindly by three different clinical laboratory technologists using two DiaMed particle gel immunoassays (ID-PaGIA) for IgA deficiency and for antibodies to IgA. The results were subsequently checked with the results of a fluorescence enzyme immunoassay conducted in the reference immunology laboratory.

Results

The ID-PaGIA had a sensitivity of 91.7% and specificity of 97.1% for the IgA deficiency test. With regards to the detection of anti-IgA antibodies, the sensitivity was 89.3% and the specificity 100%. The reproducibility of the test was 100%.

Discussion

The ID-PaGIA screening assays are suitable for the investigation of transfusion-related anaphylactic reactions in a routine blood bank laboratory. Although the gel card technique does not quantify the level of anti-IgA antibodies, it is readily available, providing an effective and simple method for the diagnosis of anti-IgA related anaphylaxis and guidance for the appropriate transfusion practice in an emergency.     

Identification of an N-terminal 27 kDa fragment of <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> P116 protein as specific immunogen in <Emphasis Type="Italic">M. pneumoniae</Emphasis>infections

Background  

Mycoplasma pneumoniae is an important cause of respiratory tract infection and is increasingly being associated with other diseases such as asthma and extra-pulmonary complications. Considerable cross-reactivity is known to exist between the whole cell antigens used in the commercial serological testing assays. Identification of specific antigens is important to eliminate the risk of cross-reactions among different related organisms. Adherence of M. pneumoniae to human epithelial cells is mediated through a well defined apical organelle to which a number of proteins such as P1, P30, P116 and HMW1-3 have been localized, and are being investigated for adhesion, gliding and immunodiagnostic purposes.     

Anti–topoisomerase I (Anti–Scl‐70) antibodies in patients with systemic lupus erythematosus

Objective

To investigate the presence and clinical significance of anti–Scl‐70 antibodies in patients with systemic lupus erythematosus (SLE).

Methods

Levels of antibodies against Scl‐70 were determined by a commercial clinical enzyme‐linked immunosorbent assay (ELISA) during routine evaluation. Results were verified by an additional ELISA with a characterized bovine Scl‐70, by ELISA with a recombinant human topoisomerase I, by Western blot, and by double diffusion in agar gel. Disease activity was estimated retrospectively by the Systemic Lupus Activity Measure (SLAM).

Results

Of 128 consecutive SLE patients, 25% were positive for anti–Scl‐70 antibody; this antibody activity was cognate in nature. No SLE patient could be classified as also having systemic sclerosis. The levels of anti–Scl‐70 were significantly correlated with the SLAM score for the entire cohort (r = 0.563, P < 0.001) and for 7 individual patients with multiple longitudinal measurements (r = 0.755–0.951, P < 0.001; n = 6) (r = 0.378, P < 0.05; n = 1). A significant correlation was also found between the levels of anti–Scl‐70 and anti–double‐stranded DNA antibodies (r = 0.558, P < 0.001). Patients with anti–Scl‐70 had significantly higher risk of pulmonary hypertension (P < 0.01) and renal involvement (P < 0.001) than patients without this antibody.

Conclusion

Anti–Scl‐70 antibody is present in a significant subset of patients with SLE. For this subset, it offers a good correlate of disease activity and suggests increased risk for pulmonary hypertension and nephritis.

     

Antibodies to guanosine triphosphate misidentified as anti–double‐stranded DNA antibodies in a patient with antinuclear antibody–negative lupus,due to buckling of insolubilized assay DNA

Objective

To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double‐stranded DNA (dsDNA) as revealed by enzyme‐linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp‐2 immunofluorescence [IF]).

Methods

The patient's antibodies were isolated on dsDNA and single‐stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp‐2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies. Other tests performed included immunoblotting, immunoprecipitation, Crithidia luciliae IF, and neutrophil IF.

Results

The antibodies isolated from the dsDNA and ssDNA supports were similar, in that they were of the IgG type, bound well in the dsDNA ELISA, and recognized a normally hidden nucleolar RNA antigen in HEp‐2 cells. With both the dsDNA ELISA and nucleolar antigens, inhibition studies revealed that the epitope recognized was guanosine 5′‐triphosphate (GTP). Binding of the antibodies was better to GTP than to guanosine 5′‐monophosphate or cytidylyl (3′‐5′) guanosine, and, in turn, was better than to guanosine, while N7‐methylated GTP was unreactive. The antibodies did not bind to dsDNA present in solution or in HEp‐2 or Crithidia cells, but bound transfer RNA well and recognized a cytoplasmic RNA antigen in neutrophils.

Conclusion

A new problem in dsDNA ELISA is revealed in the occurrence of a hitherto‐unknown and unusual buckling of the insolubilized DNA molecule, which, absent in dsDNA found in solution or in whole cells, presumably creates gaps of single‐strandedness in the molecule. A new antibody specific for GTP is described in this patient, which may be clinically important.

     

Accuracies of <Emphasis Type="Italic">Leuconostoc</Emphasis>phenotypic identification: a comparison of API systems and conventional phenotypic assays

Background  

Commercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria.     

Sensitivity of Serum Tissue Transglutaminase Antibodies for Endomysial Antibody Positive and Negative Coeliac Disease

Background: Serum antibodies to tissue transglutaminase (tTGA) are reported to have high sensitivity and specificity for coeliac disease and to correlate closely with endomysial antibodies (EmA). We assessed their performance in a coeliac population with a high proportion of EmA-negative patients, who have been under-represented in previous studies. Methods: We used a commercial ELISA kit to test for IgA class tTGA in sera from a population of 73 untreated coeliac patients with normal serum IgA and a high percentage (19%) EmA-negative, taking 58 patients with normal duodenal biopsies as controls. EmA was measured using indirect immunofluorescence. Results: Forty-six (63%) patients with villous atrophy (VA) had both tTGA and EmA. However, when considered separately, sensitivities of tTGA and EmA for VA were similar (75% versus 81%) and both had high specificity (98% versus 97%). As 9 patients were tTGA-positive only and 13 had EmA only, selection of patients for biopsy on the presence of either antibody would have had a sensitivity of 93% (68 of 73), with 5 (7%) patients seronegative for both. Conclusion: Although the ELISA tTGA assay is more convenient than EmA testing, it offers no advantages in sensitivity or specificity if used in isolation. However, incomplete concordance between EmA and tTGA positivity means that combination screening with both assays offers higher sensitivity, as almost a third of patients have only one antibody. As some coeliac patients with normal serum IgA are negative for both antibodies, biopsies should still be performed in seronegative individuals deemed at high risk for coeliac disease.     

Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania

Background  

Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from HIV-infected patients, pregnant women, voluntary counseling and testing attendees and blood donors, and to formulate an alternative confirmatory strategy based on rapid HIV testing algorithms suitable for use in Tanzania.     

Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

Background  

Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously.     

Laboratory detection of strongyloidiasis: IgG‐, IgG4‐ and IgE‐ELISAs and cross‐reactivity with lymphatic filariasis

Enzyme‐linked immunosorbent assays (ELISAs) were developed for the detection of IgG, IgG₄ and IgE antibodies against Strongyloides stercoralis. A commercial ELISA (IVD Research, USA) was also used, and the sensitivities and specificities of the four assays were determined. Serum samples from 26 patients with S. stercoralis infection and 55 patients with other infections or no infection were analysed. Sensitivities of the IgG₄, IgG, IgE and IgG (IVD) assays were 76·9%, 84·6%, 7·7% and 84·6%, respectively, while the specificities were 92·7%, 81·8%, 100% and 83·6%, respectively. If filariasis samples were excluded, the specificities of the IgG₄‐ELISA and both IgG‐ELISAs increased to 100% and 98%, respectively. A significant positive correlation was observed between IgG‐ and IgG₄‐ELISAs (r = 0·4828; P = 0·0125). IgG‐ and IgG‐ (IVD) ELISAs (r = 0·309) were positively correlated, but was not significant (P = 0·124). Meanwhile there was no correlation between IgG₄‐ and IgG‐ (IVD) ELISAs (r = 0·0042; P = 0·8294). Sera from brugian filariasis patients showed weak, positive correlation between the titres of antifilarial IgG₄ and the optical densities of anti‐Strongyloides IgG₄‐ELISA (r = 0·4544, P = 0·0294). In conclusion, the detection of both anti‐Strongyloides IgG₄ and IgG antibodies could improve the serodiagnosis of human strongyloidiasis. Furthermore, patients from lymphatic filariasis endemic areas who are serologically diagnosed with strongyloidiasis should also be tested for filariasis.     

Advances in serological,imaging techniques and molecular diagnosis of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> infection

Background

Toxoplasmosis is worldwide distributed zoonotic infection disease with medical importance in immunocompromised patients, pregnant women and congenitally infected newborns. Having basic information on the traditional and new developed methods is essential for general physicians and infectious disease specialists for choosing a suitable diagnostic approach for rapid and accurate diagnosis of the disease and, consequently, timely and effective treatment.

Methods

We conducted English literature searches in PubMed from 1989 to 2016 using relevant keywords and summarized the recent advances in diagnosis of toxoplasmosis.

Results

Enzyme-linked immunosorbent assay (ELISA) was most used method in past century. Recently advanced ELISA-based methods including chemiluminescence assays (CLIA), enzyme-linked fluorescence assay (ELFA), immunochromatographic test (ICT), serum IgG avidity test and immunosorbent agglutination assays (ISAGA) have shown high sensitivity and specificity. Recent studies using recombinant or chimeric antigens and multiepitope peptides method demonstrated very promising results to development of new strategies capable of discriminating recently acquired infections from chronic infection. Real-time PCR and loop-mediated isothermal amplification (LAMP) are two recently developed PCR-based methods with high sensitivity and specificity and could be useful to early diagnosis of infection. Computed tomography, magnetic resonance imaging, nuclear imaging and ultrasonography could be useful, although their results might be not specific alone.

Conclusion

This review provides a summary of recent developed methods and also attempts to improve their sensitivity for diagnosis of toxoplasmosis. Serology, molecular and imaging technologies each has their own advantages and limitations which can certainly achieve definitive diagnosis of toxoplasmosis by combining these diagnostic techniques.

     

Prevalence of celiac disease and celiac autoimmunity in the Toba native Amerindian community of Argentina

BACKGROUND:

Celiac disease (CD) is mostly recognized among subjects with a Caucasian ethnic ancestry. No studies have explored conditions predisposing Amerindians to CD.

OBJECTIVE:

To prospectively assess environmental, genetic and serological conditions associated with CD among members of the Toba native population attending a multidisciplinary sanitary mission.

METHODS:

An expert nutritionist determined daily gluten intake using an established questionnaire. Gene typing for the human leukocyte antigen (HLA) class II alleles was performed on DNA extracted from peripheral blood (HLA DQ2/DQ8 haplotype). Serum antibodies were immunoglobulin (Ig) A tissue transglutaminase (tTG) and the composite deamidated gliadin peptides/tTG Screen test. Positive cases were tested for IgA endomysial antibodies.

RESULTS:

A total of 144 subjects (55% female) were screened. The estimated mean gluten consumption was 43 g/day (range 3 g/day to 185 g/day). Genetic typing showed that 73 of 144 (50.7%) subjects had alleles associated with CD; 69 (94.5%) of these subjects had alleles for HLA DQ8 and four had DQ2 (5.5%). Four and six subjects had antibody concentrations above the cut-off established by the authors’ laboratory (>3 times the upper limit of normal) for IgA tTG and deamidated gliadin peptides/tTG screen, respectively. Four of these had concomitant positivity for both assays and endomysial antibodies were positive in three subjects who also presented a predisposing haplotype.

CONCLUSION:

The present study was the first to detect CD in Amerindians. The native Toba ethnic population has very high daily gluten consumption and a predisposing genetic background. We detected subjects with persistent CD autoimmunity and, at least, three of them fulfilled serological criteria for CD diagnosis.     

Assessment of factors associated with smoking cessation at diagnosis or during follow‐up of Crohn's disease

Background and Aim

Smoking cessation is known to improve the course of Crohn's disease (CD). However, the factors associated with smoking cessation after CD diagnosis have not been well established.

Methods

Clinical characteristics and change in smoking status were evaluated in 445 current smokers at the time of CD diagnosis. Patients were classified into three subgroups based on their final smoking status and time of smoking cessation: non‐quitters, quitters at diagnosis, and quitters during follow‐up.

Results

The overall smoking cessation rate was 55.7% (248 of 445 patients). The diagnosis of CD was the main reason for quitting (41.5%, 103 of 248 patients). Smoking cessation at the time of CD diagnosis was associated with intestinal resection within 3 months from CD diagnosis (odds ratio [OR] 2.355, 95% confidence interval [CI] 1.348–4.116, P = 0.003), light smoking (OR 2.041, 95% CI 1.157–3.602, P = 0.014), and initiation of smoking before 18 years of age (OR 0.570, 95% CI 0.327–0.994, P = 0.047). Light smoking (OR 1.762, 95% CI 1.019–3.144, P = 0.043) and initiation of smoking before 18 years (OR 0.588, 95% CI 0.381–0.908, P = 0.017) were also associated with overall smoking cessation.

Conclusion

Quitters after CD diagnosis, including quitters at diagnosis and quitters during follow‐up, had features distinct from those of non‐quitters. Given the motivation at CD diagnosis, a detailed history of smoking habits should be taken and all current smokers should be encouraged to quit smoking at the time of CD diagnosis.