Occurrence Of False-positive Results Research Articles

Testing the specificity of the diatom test: search for false-positives

The diatom test is widely used by forensic pathologists as proof of drowning, notwithstanding some criticisms mainly concerning the occurrence of false-positive results (presence of diatoms in the tissues of subjects who died from causes other than drowning). The aim of the present study was to verify the claimed inaccuracy of the method caused by an excessive rate of false-positives related to inadvertent exposure to diatoms of the general population. The study was carried out to investigate the presence of diatoms in the tissues (lungs and sternum) of subjects who died from causes other than drowning. Two groups of cadavers that underwent an autopsy at the Institute of Forensic Medicine of the University of Verona were included in the study. Group A comprised 45 individuals who died from causes other than drowning, whereas Group B comprised 20 bodies which had been recovered from water. The extraction of the diatoms was performed by incubation of samples in nitric acid for 48 hours at 60°C. The analysis of the samples from Group A showed the absence of diatoms in both lung and sternum samples. In Group B all lung samples showed the presence of diatoms, whereas only six sternum samples were shown to contain diatoms. The difference between Groups A and B was statistically highly significant. The absence of diatoms in the samples collected from Group A falsified the hypothesis that false-positive results from the diatom test may occur due to diatoms entering living bodies through the respiratory and/or digestive tracts via air, water or food, supporting the validity of the diatom test as proof of drowning.

Occurrence of false-positive results in three Paratuberculosis - ELISAs performed in a tuberculous herd

The interference of bovine tuberculosis (TB) on the efficacy of paratuberculosis (PTB) diagnostic tests has been evaluated. A group of 32 tuberculous cows identified by both intradermal tests and gamma-interferon assay, 16 of them confirmed by the recovery of M.bovis from tissues, was tested by three different PTB- ELISAs, being two commercials and one in-house. The rest of the adult animals of the herds, totalizing 216 TB-negative animals, were also tested as a control group. Fecal culture for PTB was negative in all animals, but seven (21.8%) tuberculous cows produced false-positive reactions when tested by various PTB-ELISAs, leading to a misdiagnosis. Tuberculosis impairs the specificity of serological tests for paratuberculosis diagnosis and should be considered for the reliability of PTB control programs.

Circulating tumour cells in breast cancer: Prognostic indicators, metastatic intermediates, or irrelevant bystanders? (Review)

Circulating tumour cells (CTCs) have been of considerable interest for many years. The rarity of these cells presents the main challenge associated with their analysis. Current detection methods use antibody and nucleic acid techniques and are sensitive for CTC detection but limited in their utility by the occurrence of false-positive results. Despite this, there are a number of clinical studies which show that the presence of CTCs is an important prognostic indicator, particularly in the metastatic setting. Current efforts to phenotype CTCs may provide a valuable insight into the metastatic process and may also allow the development of specific CTC-targeted treatment strategies in the future.

Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification

A novel method for regenerating biosensors has been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems requiring low power consumption such as remote field sensing applications. Gold-sputtered 96-well polystyrene microplates and a fluorescent label were used to explore the detection limits of the surface-associated RCA technique, specificity for different MPP, conditions for regeneration of the biomolecular sensing surface, and reproducibility of measurements on regenerated surfaces. The technique was used to create highly selective biomolecular surfaces capable of discriminating between DNA oligonucleotides with sequences identical to RNA from infectious salmon anemia (ISA) and infectious hematopoietic necrosis (IHN) virus. As little as 0.6 fmol of circularized MPP was detectable with this fluorimetric assay. The sensing layers could be reused for at least four cycles of amplification using thermal denaturation, with less than 33% decrease in RCA response over time. Because the nucleic acid product of the test is attached to a surface during amplification, the technique is directly applicable to a variety of existing sensing platforms, including acoustic wave and optical devices.

Anticardiolipin, other antiphospholipid antibody tests and diagnosis of the antiphospholipid syndrome

The frequent occurrence of false positive results in the anticardiolipin (aCL) enzyme linked immunosorbent assay (ELISA) hampers its application in identifying the antiphospholipid syndrome (APS), a condition characterized by a myriad of clinical presentations. This study highlights some of the pitfalls in the use of assays for antiphospholipid (aPL) antibody in clinical practice. The aCL ELISA, commercially prepared anti-beta2-gylcoprotein 1 (beta2-GP1) and antiphospholipid (APhL) assays were evaluated in the diagnosis of antiphospholipid syndrome (APS) in 94 pregnant women who had spontaneous abortion and a group of 177 healthy blood donors. Serological tests were used to rule out syphilis as the cause of false positive results in the aCL ELISA. The prevalences of positive aCL ELISA results (29/94, 31% v 26/177, 14%; p = 0.001) and aCL antibodies of the IgM isotype (19/94, 20% v 6/177, 3%; $p = 0.001$) were significantly higher in aborters compared to healthy subjects. The majority of the sera which were positive in the aCL ELISA were shown to be false positives as 93% (27/29) of aCL positive aborters and 67% (8/24) of aCL positive healthy subjects were negative in the anti-beta2-GP1 assay. Similarly, the sensitivity of the APhL ELISA was low and only 1% (1/94) of the sera of aborters and 6% (11/177) of healthy subjects were positive in this assay. The frequent occurrence of anticardiolipin antibodies of the authentic non-autoimmune variety and the low sensitivity of the other more specific aPL assays make the positive aCL ELISA difficult to interpret. We recommend that the diagnosis APS be made with strict adherence to the preliminary criteria for classification of APS.

Comparison of culture, polymerase chain reaction (PCR), TaqMan Salmonella, and Transia Card Salmonella assays for detection of Salmonella spp. in naturally-contaminated ground chicken, ground turkey, and ground beef

Four types of assays were evaluated for the detection of Salmonella spp. in retail ground chicken (86 packages), ground turkey (104 packages), and ground beef (54 packages). Two 25 g samples from each package were separately subjected to pre-enrichment in buffered peptone water for 20 h at 37 °C followed by enrichment in Rappaport Vassiliadis (RV) broth for 20 h at 42 °C. The RV enrichments were plated onto Rambach agar, Rainbow Agar Salmonella, and XLT4 agar, and were also tested by a PCR assay targeting the Salmonella invA gene, as well as by the TaqMan ® Salmonella PCR assay. Additionally, the RV enrichments were tested using the Transia Card Salmonella ® immunoassay. Results showed that 16.8, 24.0, 28.8, and 26.4% of turkey samples were positive for Salmonella spp. by culture, PCR, TaqMan PCR, and Transia Card Salmonella assays, respectively. Eighteen, 28.5, 35.5, and 34.9% of chicken samples were positive by culture, PCR, TaqMan PCR, and Transia Card Salmonella assays, respectively, and 6.5, 6.5, 6.5, and 18.5% of ground beef samples were positive by the four assays, respectively. Analysis of the data using the kappa statistic showed that there was substantial to excellent agreement between the PCR and TaqMan PCR assays and between the PCR and culture assays (kappa coefficients ranging from 0.67 to 0.87), while there was poor to fair agreement between the results of the Transia Card Salmonella assay and the other methods (kappa coefficients ranging from 0.28 to 0.32). Overall, results showed that the PCR-based assays were more sensitive than the culture method, and the culture and PCR-based assays were more specific than the immunoassay for detection of Salmonella in ground chicken, turkey, and beef due to the occurrence of false positive results using the immunoassay.

False-positive results in neuroblastoma screening: the parents' view.

One of the major problems of any screening program is the occurrence of false-positive results. For neuroblastoma screening, little information is available on the psychological consequences for parents whose children had false-positive results that made further clinical evaluation necessary. It was the aim of this study to evaluate the parents' view by a semistructured interview. Among 267,302 infants screened in the Austrian study between 1991 and 1999, 19 had to be considered as repeatedly false-positive (no clinical evidence of neuroblastoma). Sixteen of 19 parent pairs could be reached by phone and were interviewed separately by use of a semistructured questionnaire to evaluate for psychological consequences resulting from the screening result. The psychological burden appeared to be small during the initial screening procedure, but it increased significantly through hospital admission and was then described as severe by 19 of the 32 parents. Investigators should be aware of the psychological consequences of hospital admission for tumor screening in children. In ongoing neuroblastoma screening studies, laboratory methods as well as cutoff limits should be selected carefully.

Interference of the Position of Substances in an Epicutaneous Patch Test Battery With the Occurrence of False-Positive Results

Interference of the Position of Substances in an Epicutaneous Patch Test Battery With the Occurrence of False-Positive Results

An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis

A cocktail sandwich ELISA based on the employ of two monoclonal antibodies (MAbs) as coating antibodies and a third MAb conjugated to horseradish peroxidase has been developed for the analysis of gluten in foods. Given that each MAb displays a wide specificity spectrum for wheat, barley, rye and oats prolamins, their combination for ELISA ensures a high crossreactivity with most of the potentially toxic gliadin, hordein, secalin and avenin protein family. One of the unprecedented features of the cocktail sandwich ELISA is that it permits for the first time analysis of barley hordeins in foods, which is unattainable using conventional or commercial ELISA kits. Besides, gliadins, hordeins and secalins are recognised to the same extent. The system provides a high detection sensitivity for gliadins, hordeins, secalins and avenins (1.5, 0.05, 0.15 and 12 ng/ml, respectively). The working linear range comprises 3–100 ng/ml with a gliadin detection limit of 1.5 ppm. This limit of detection is even better than that demanded in the latest Codex recommendation, 10 ppm. Cocktail ELISA data were contrasted with those of commercial ELISA kits and confirmed by mass spectrometry, a non-immunological technique which provides evidence for the occurrence of false positive results with the commercial kits.

Contamination-free and automated composition of a reaction mixture for nucleic acid amplification using a capillary electrophoresis apparatus

The acceptance of the polymerase chain reaction (PCR) as an amplification method in molecular diagnostics and the rapid development of capillary electrophoresis (CE) as an analysis method of those PCR products was a reason for us to investigate further integration of those two techniques. Using a fused-silica capillary as a pipette we were able to compose a PCR mixture in the CE apparatus. Because a capillary can be thoroughly rinsed and the CE apparatus is a closed system, the risk of contamination and therefore the occurrence of false positive results is minimized. The fact that a CE system can be fully automated contributes to a more reproducible and standardized PCR composition protocol.

Detection of Aspergillus galactomannan antigen in foods and antibiotics

The specificity of the Pastorex Aspergillus latex agglutination test for the diagnosis of manifest aspergillosis is hampered by the occurrence of false-positive results. In order to prove whether or not the false-positive reactions may be caused by the uptake of the soluble galactomannan antigen from the environment, the presence of the antigen was tested in foods, air samples, antibiotics for therapeutic use and faeces. Reactions of the Aspergillus latex agglutination test were found in 15 (79%) out of 19 samples of meals prepared in a hospital kitchen, in five out of six canned vegetables from a supermarket, in all of six samples of pasta and rice bought in health shops, in the faeces of four bone marrow transplant (BMT) recipients and of four healthy subjects and in one and two batches of the antibiotics co-amoxyclav and piperacillin respectively. The concentration of the antigen in faecal material was calculated to be in the range of 1.2-38.4 micrograms g-1. It is concluded that the faecal galactomannan antigen may reach the circulation in patients with dysfunction of the intestinal mucosal barrier, e.g. BMT recipients, thus leading to diagnostically false-positive antigenaemia.

Diagnostic potential of a serological assay for the diagnosis of ulcerans disease based on the putative Mycobacterium ulcerans toxin.

Mycobacterium ulcerans infection is an important and potentially disfiguring disease of man. A rapid diagnostic assay for detection of this organism is required urgently. Serological assays require a species-specific protein to ensure a high level of specificity and thus reduce the occurrence of false positive results. As M. ulcerans had been reported to produce a unique cytotoxin, it was thought that this would provide an ideal antigen on which to base a serological assay for detection of M. ulcerans during infection. Crude culture filtrates, prepared by previously documented methods, were assayed for toxic activity by in-vitro cytotoxicity assays and in-vivo mouse footpad assays. To evaluate the uniqueness of the cytotoxic factor, other species of mycobacteria were also assayed. Analysis of these assays showed that similar biological activity is present in various other mycobacterial species. Furthermore, it was possible to neutralise this activity in all species tested with a polyclonal antiserum raised against M. ulcerans. As the cytotoxic factor was found not to be specific to M. ulcerans, it is unlikely that a serological assay based on such a molecule will be of use.

Hepatitis E virus seroprevalence in acute viral hepatitis in a developed country confirmed by a supplemental assay.

Hepatitis E virus (HEV) infection is prevalent among cases of acute viral hepatitis in young adults in developing countries. HEV infection is not restricted to endemic areas, but would appear to be worldwide in distribution. In order to document the incidence of HEV infection in acute hepatitis cases in a developed country, IgG and IgM anti-HEV antibodies and HEV RNA were tested in 101 Caucasian patients with acute viral hepatitis; 92 of these cases had markers of acute viral hepatitis other than HEV. Forty-seven (46.5%) cases had IgG anti-HEV; IgM anti-HEV and HEV viremia were not detected. As the incidence of anti-HEV was higher than would be expected, the possibility of the occurrence of false positive results was subsequently investigated. Supplemental antibody testing, using a broadly reactive epitope region, reduced the frequency of anti-HEV to 17%. Therefore, supplemental antibody testing confirms the hepatitis E virus seroprevalence in a developed country. Since IgM anti-HEV and HEV viremia were not detected, persons with IgG anti-HEV may be "subclinical HEV cases," or have long-lived antibodies in their circulation.

Evaluation of tissue polypeptide antigen serum levels for monitoring disease activity during chemotherapy in patients with transitional carcinoma of the urinary tract

In 28 patients with transitional carcinoma of the urinary tract, all treated with chemotherapy, serial measurements of serum tissue polypeptide antigen (TPA) were performed and correlated to clinical evaluations of response. At the start of chemotherapy elevated levels of TPA were found in 4 out of 14 patients with T2-4NO-2MO tumours and in 7 out of 14 patients with distant metastases. In most patients with elevated TPA levels who responded to chemotherapy, TPA levels rapidly returned to normal. False positive elevations of TPA were observed in 2 patients. It is concluded that serial measurement of TPA for monitoring disease activity has limited value because of the low sensitivity of TPA, especially for patients with early-stage cancer, and because of the occurrence of false positive results.

Assay for Antibody in Pig Fetuses Infected with Porcine Parvovirus

Fetal fluids from field cases of fetal death were assayed for antibody to porcine parvovirus (PPV) using 3 different techniques. An indirect immunofluorescent antibody test, a counter immunoelectrophoresis test and a hemagglutination inhibition test were compared. The indirect immunofluorescent antibody test was found to be the most sensitive of the tests employed. The hemagglutination inhibition test apparently suffered from the occurrence of false positive results.

Otitis media with effusion: Accuracy of tympanometry in detecting fluid in the middle ears of children at myringotomies.

Objective:(1) The diagnostic accuracy of tympanometry in detecting fluid in the middle ear space in children with otitis media with effusion by comparing its findings with those of myringotomies. (2) Identify the age group most commonly affected by OME.Methods:This prospective study was conducted at the Department of ENT& Head and Neck Surgery, Postgraduate Medical Institute Hayatabad Medical complex, Peshawar from July 1, 2012 to April 30, 2015. Patients with suspicion of OME underwent tympanometry and later myringotomies. Using Jerger’s classification, Type B tympanogram with normal canal volume was considered as conclusive evidence of fluid in the middle ear space. Its findings were compared with those of the respective myringotomies. From the data collected, the accuracy, sensitivity, specificity, positive predictive value and negative predictive values were calculated.Results:A total 117 ears of 63 patients were operated. The age range was 3 to 12 years. The commonest age group (58.7%) affected by OME was 6-8 years. Type B tympanogram with flat curve and normal canal volume was obtained in 71.4% of the ears. Comparison with myringotomy findings showed TP 85, TN 13, FP 5 and FN 14. The diagnostic value of tympanometry was; Sensitivity 85.85%, Specificity 72.22%, PPV 94.44%, NPV 48.14% and Accuracy of 83.76%. P value calculated using chi square test showed that there was significant difference between tympanometry and myringotomy findings in OME (p < 0.05).Conclusions:OME is common in age group 6-8 years. Tympanogram Type B with normal canal volume is fairly sensitive in diagnosing this condition. However for occurrence of false positive results, final decision regarding management should be made on clinical findings and other supportive audiological tests.