Cause Of False Positive Reactions Research Articles

Performance of nucleocapsid and spike-based SARS-CoV-2 serologic assays.

There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance along with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the probable causes of false-positive reactions were determined. The assays were evaluated using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of false positive results from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, assays lacked reproducibility. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with the same antigen that was used for coating ELISAs prior to performing the assay), suggesting positive reaction could be attributed to the presence of antibodies against these antigens. In other cases, reactivity was consistently detected but not abrogated by the spiking, suggesting positive reaction was not attributed to the presence of antibodies against these antigens. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of “false positivity” in pre-epidemic samples may be due to plasma antibodies apparently reacting with the corresponding antigen, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

Intercurrence of Paratuberculosis in Intradermal Tuberculin Test Reactive Cattle

Background: Bovine tuberculosis control programs are based on a standard diagnostic method, the intradermal test with purified protein derivatives, which is used to identify and eliminate diseased animals. Currently none of the tests available allow complete differentiation between infected and uninfected animals. The main limitations of the tests available are related to diagnostic sensitivity and specificity, which results in false positive reactions due to the existence of cross infections, and also false negative, inherent to the state of energy of some animals. The aim of this work was to study the intercurrence of paratuberculosis in tuberculosis reactive cattle by the comparative cervical test.Materials, Methods & Results: Three hundred and thirty four cattle were evaluated using the comparative cervical test (CCT) and serology for tuberculosis (TB) and paratuberculosis (PTB) ELISA IDEXX®. All of the animals testing positive by CCT were euthanized and necropsied. Fragments of lymph node, lung and intestine were collected and analyzed using histopathological techniques, with staining by Hematoxylin-Eosin (HE). Samples of lung and lymph nodes (retropharyngeal, submandibular, cervical and mediastinal) of the animals testing positive by CCT were evaluated using qPRC for M. bovis, and intestinal and mesenteric lymph nodes using PCR for PTB. Of the 334 cattle evaluated using the comparative cervical test, 16 were considered positive. No lesions suggestive of tuberculosis were found in the macroscopic inspection of the carcasses. The most evident anatomical and pathological finding was a thickening of intestinal mucosa, found in 12 of the 16 cattle submitted to necropsy. No microscopic lesions suggestive of TB were identified nor was the presence of M. bovis detected by qPCR. The main histopathological findings were observed in the small intestine and mesenteric lymph nodes and identified as enteritis, lymphangitis, lymphangiectasia and granulomatous lymphadenitis. In the intestine the changes are characterized by dilated and inflamed lymphatic vessels and intense inflammatory infiltrate on the mucosa and submucosa. Of the 334 serum samples evaluated, the M. bovis ELISA Antibody Test (IDEXX®) identified 17 positive animals. All the cattle considered positive by M. bovis ELISA were considered negative by CCT. In the samples from nine animals (9/16), DNA from M. avium subsp. paratuberculosis (MAP) was identified and in twelve carcasses (12/16) lesions characteristic of PTB were found, which were subsequently confirmed by histopathological techniques. In another nine animals of the herd anti-MAP antibodies were detected. None of those that tested positive by PTB ELISA were reactive by CCT.Discussion: Animals considered positive by TB ELISA that were not positive in the intradermal test does not mischaracterize the clinical picture of the disease. Considering the inverse relationship between cell-mediated and humoral responses to M. bovis, the intradermal test and the serological tests are designed to measure different immunological responses, which develop during different stages of infection. The progress of the cellular immunological response to humoral immunity occurs in the most advanced stages of tuberculosis. Of the 16 cattle considered positive by CCT, 12 animals presented macroscopic and histological lesions suggestive of PTB and DNA from MAP was detected in nine. Although it is the official test for the control of TB in different countries, the intradermal test with PPD has presented limitations, primarily related to specificity. M. avium subsp. Paratuberculosis is considered the main cause of false positive reactions in the intradermal test. The PPD bacterial extract is a complex mixture of proteins, lipids, sugars and nucleic acids, and many of these components are also shared by numerous species of mycobacteria (tuberculous or not).

Effect of paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed infection using interferon-gamma detection assay

Interferon-gamma (IFN-γ) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-γ assay was studied in a bullfighting cattle herd with a mixed tuberculosis–paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-γ assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-γ detection test.

Monitoring of dairy herds for Brucella abortus infection when prevalence is low.

A total of 2,698 dairy herds were surveyed in 1981–1982 in New South Wales and north eastern Victoria in a review of the methods used to monitor them for the presence of Brucella abortus., The methods used to monitor dairy herds were testing of all breeding cows over 1 year of age using the rose bengal test (RBT) and complement fixation test (CFT), the bulk milk ring test (BMRT), and testing of blood samples collected at abattoirs using the RBT and CFT. The surveyed herds had at least one whole herd test, and BMRT was done at regular intervals in the period of the survey.Of the 99 (3.7%) herds that reacted to the BMRT, 91 (3.4%) herds had false positive reactions and 8 (0.3%) herds were declared infected on follow‐up herd testing. False‐positive reactions were obtained in 22 herds on more than one occasion. Common causes of false positive reactions to the BMRT were thought to be previous vaccination with Strain 19 and sampling in very early or late lactation.Of the 98 (3.63%) herds that reacted to the whole herd serological tests, 80 (2.96%) herds had false‐positive reactions and 18 (0.67%) herds were declared infected. Strain 19 vaccination was thought to be an important cause of false‐positive reactions. Fifty‐three (2.0%) herds showed suspicious reactions on abattoir monitoring but none was declared infected on follow‐up testing.Of the 18 herds with infected or equivocal status, the BMRT identified 8. In a further 6 herds, the infected cattle were not in the milking herd. Four other herds had milkers with high CFT titres which could not be confirmed as infected on culture. In no herds were culture positive RBT or CFT reactors from the milking herd detected without the BMRT being positive. The proportion of false‐positive reactions to the BMRT was high but the BMRT proved very useful in identifying dairy herds infected with B. abortus, when the prevalence of brucellosis was very low. Aust Vet J, 64: 97–100

HLA antibodies as a cause of false-positive reactions in screening enzyme immunoassays for antibodies to human T-lymphotropic virus type III.

15,680 volunteer blood donors were screened by enzyme-linked immunosorbent assay (EIA) for antibodies to human T-lymphotropic virus Type III (HTLV-III). On at least two of three determinations, 0.37 percent were found to be reactive. When the first 38 of these samples were sent for repeat EIA and Western blot, the EIA was positive in 14. In only three of these 14 samples was the EIA result confirmed by Western blot. Eight of these eleven false-positive samples were from women who were found to have HLA antibodies. In seven of these women, the HLA antibodies were directed to Class II antigens expressed on the cell used to grow HTLV-III in the preparation of screening EIA kits. We conclude that HLA antibodies are an important cause of false-positive reactions in the screening test for HTLV-III antibody.

Rheumatoid factor-like reactants in sera proven to transmit non-A, non-B hepatitis: a potential source of false-positive reactions in non-A, non-B assays.

Convalescent phase non-A, non-B (NANB) human and chimpanzee sera were utilized in a solid-phase radioimmunoassay (RIA) in an attempt to identify a specific NANB antigen in human plasma, plasma-derived pellets and NP-40 disrupted pellets proven to transmit NANB infection to chimpanzees. RIA reactivity was noted in 5 of 8 pedigreed NANB infectious plasmas, but did not appear to be virus-specific. The RIA reactive fraction: (i) had a density of 1.27 to 1.32 gm per cm3 in cesium chloride; (ii) showed immunoreactivity corresponding to normal IgM, but peak RIA activity coincided with an aberrant IgM having a peak sedimentation coefficient of 56 S, and (iii) eluted at higher pH (5.6 to 5.0) than normal IgM on chromatofocusing columns. The reactant shared properties with rheumatoid factor in that it was absorbed by aggregated IgG and was unstable to 2-mercaptoethanol, but it did not react in standard agglutination tests for rheumatoid factor. The reactant was detected in both the acute and chronic phase of NANB infections in chimpanzee and man. By reacting with IgG in presumptive NANB convalescent sera, this aberrant rheumatoid factor could simulate a NANB antigen and represent a cause of false-positive reactions in putative NANB assays.

Stool desorbing activity: a possible cause of false-positive reactions in competitive enzyme immunoassays.

We have developed a competitive enzyme immunoassay for the measurement of purified toxin A of Clostridium difficile. However, when we applied this assay to the detection of C. difficile toxin in stool specimens, we noted a high rate of nonspecific activity in fecal specimens which did not contain toxin. We found that the low specificity (26%) of the assay was due to the presence in stool specimens of interfering factors which desorbed the antigen coated on the solid-phase surface. These factors could be detected by measurement of the desorption of biotin-labeled proteins attached to the solid-phase surface. In addition, these interfering factors were partially inactivated by heating at 56 degrees C for 10 min and partially inhibited by phenylmethylsulfonyl fluoride (2 mM) or soybean trypsin inhibitor (10 mg/ml). These data suggested that the desorbing activity was due to proteolytic activity in the fecal specimens. Fetal calf serum (50%) was found to be the most effective measure in preventing the interfering effect. By using 50% fetal calf serum as a diluent, we increased the specificity of the antibody inhibition enzyme immunoassay to 93%. Interfering factors in stool specimens could be a cause of false-positive results in other competitive immunoassay systems. The use of diluents which neutralize protease activity can result in a marked improvement in the specificity of competitive immunoassay systems.

False Positive and False Negative Reactions in HLA B‐27 Antigen Testing

The recent commercial availability of HLA B-27 antiserum now enables laboratories without prior experience in HLA typing to test for this antigen. False positive and false negative reactions frequently occur and require judicious interpretation. In our laboratory, 30 of 100 specimens were positive for B-27 and 70 were negative. Of the 30 positive, 10 (33%) failed to react against every antiserum tested. Twenty-nine of the 70 negative specimens (41%) reacted falsely with one or more B-27 antisera. A significant cause of false positive reactions was cross-reaction of the B-7 antigen with B-27 antisera (13 of 15 B-7 positive, B-27 negative specimens). For valid B-27 testing, use of more than one B-27 antiserum is included.

Rubella hemagglutination-inhibition test: false-positive reactions in sera contaminated with bacteria.

We have shown that bacterial contamination of sera can have a marked influence on the results of the rubella hemagglutination-inhibition (HI) test. In addition to increasing the levels of nonspecific agglutinins, a number of common bacterial species tested had a significant effect on the HI titers. When sera free from rubella-specific antibodies were contaminated with Bacillus subtilis or Pseudomonas fluorescens, HI titers ranged from 16 to 128 (expressed as the reciprocal of the highest serum dilution completely inhibiting hemagglutination) after treatment with heparin/MnCl2. Our observations demonstrate, therefore, that bacterial contamination can be one of the causes of false-positive reactions in this test.

Heterotypic antibodies in Liberian sera causing anomalous reactions when using a commercial haemagglutination test for hepatitis-B surface antigen.

Agglutinins reacting with normal and tanned sheep erythrocytes were the probable cause of false positive reactions given by 51 of 214 Liberian sera when using a commercial passive-haemagglutination test for hepatitis-B surface antigen. Absorption showed these agglutinins to be identical to those described earlier in Nigerian sera. Rheumatoid factor and anti-sheep-serum antibodies although present in 12 and five per cent respectively of all sera were not responsible for any false positive reactions. The practical conclusion is that such tests, based on sheep erythrocytes are unsuitable for screening this population.

The species distribution of three concanavalin A-purified mycobacterial polysaccharides.

Culture filtrates prepared from 12 species of mycobacteria were examined for the presence of 3 mycobacterial polysaccharides previously purified by concanavalin A affinity chromatography. Four commercial Old Tuberculins, tuberculin tine test units, and purified protein derivative were also studied. All 3 polysaccharides were demonstrated in all of the filtrates and each of the Old Tuberculins and purified protein derivative. The amount of concanavalin A-nonreactive polysaccharide was measured in clinical skin testing antigens and found to be in the range of potential significance as a cause of false positive reactions to Old Tuberculin.

Yeasts as a cause of false-positive reactions in patch tests for allergy to denture materials.

SUMMARY. The authors performed patch tests with denture materials in patients with denture sore mouth. In the tests, the patient's own saliva was used as wetting agent. In 2 series of control tests another saliva and distilled water were used to moisten the test material. Dermatitic reactions developed regularly on those test sites moistened with the patient's own saliva, if the saliva contained yeasts. This dermatitis is clinically and histologically identical with experimental candidiasis on healthy skin. It is concluded that the use of infected saliva in such tests gives rise to false positive reactions. Denture sore mouth appears to be the result of the combination of occlusion by the denture and of yeast infection rather than of yeast infection alone.

New technique for cutaneous testing: Preliminary report

W E believe that this new method wili become widely generalized for the following reasons: It is very convenient and economical, much more so than the two procedures now in use for the diagnosis of allergy, i.e., by scarifications and endermal injections. (The cost of syringes, etc., and their sterilization is eliminated.) It saves time, the results are fairly exact (within the limits of similar allergic tests), and, last but not least, it is less painful. The contamination of the antigens with traces of other antigens in endermal testing is well known as the cause of false positive reactions. This never occurs with the proposed new technique. This new method does not require the employment of adhesive tape or any patch to protect the zone of the skin that has received the allergenic extract. In this it differs from the Vollmer patch test for tuberculin.

Contamination of antigens with traces of other antigens as a cause of false positive reactions in intradermal testing

1. 1. Intradermal skin reactions in sensitive persons to control solution handled in syringes, needles, or bottles which had been deliberately contaminated with selected allergens and then rinsed and sterilized were compared with reactions to dilutions of the same allergens to give a roughly quantitative measure of each as a source of contamination. In most cases reactions as large or larger than those with 1:1,000,000 solutions were obtained. 2. 2. Allergic persons capable of reacting to 1:1,000,000 extracts by intradermal test are not rate, and an occasional patient will react to one-thousandth of this strength. 3. 3. Many allergens form stable films on glass or metal equipment which are not removed by rinsing or destroyed by sterilization. 4. 4. Cleansing methods should be proved to be adequate by skin tests on sensitive persons. Adequate, practical methods are described.

FALSE POSITIVE SEROLOGIC REACTIONS FOR SYPHILIS

In the past few years there has been a renewal of interest in false positive serologic reactions. Partly because of the increased number of tests performed as a routine on apparently nonsyphilitic persons, there is a better realization of the number of causes of false positive reactions. There are three main groups of such reactions: (1) those due to technical error, a relatively infrequent occurrence; (2) those reactions encountered in persistently false positive human serums resulting from the presence of a reagin-like component which has been demonstrated in various animal serums;¹(3) those positive serologic reactions which are found in patients with organic disease other than syphilis; they occur regularly in yaws, fairly constantly in leprosy and malaria and less frequently in many other diseases.²Further information and conclusive identification of those diseases causing positive reactions are particularly of clinical interest when the reactions are strongly positive and