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Contribution of (1->3)-beta-D-glucan chromogenic assay to diagnosis and therapeutic monitoring of invasive aspergillosis in neutropenic adult patients: a comparison with serial screening for circulating galactomannan.

Two noninvasive diagnostic tests, (1-->3)-beta-D-glucan (BG) (Glucatell) and galactomannan (GM) (Platelia Aspergillus), were used retrospectively in a twice-weekly screening for the diagnosis of invasive aspergillosis (IA) in 40 treatment episodes (one hospital visit per patient) in 40 neutropenic adult patients at high risk for IA. Five proven IA cases, three probable IA cases, and three possible IA cases were diagnosed. Diagnostic levels of both BG and GM were detected in 100% of patients with proven IA cases and in 66% of patients with probable IA cases. The kinetics of both markers in patients with IA were similar. The sensitivity, specificity, and positive and negative predictive values for GM and BG were identical, namely, 87.5, 89.6, 70, and 96.3%, respectively. False-positive reactions occurred at a rate of 10.3% in both tests, but the patients showing false-positive results were different in each test. Both tests anticipated the clinical diagnosis, computed tomography abnormalities, and the initiation of antifungal therapy in most patients, but BG tended to become positive earlier than GM. A combination of the two tests improved the specificity (to 100%) and positive predictive value (to 100%) of each individual test without affecting the sensitivity and negative predictive values. In conclusion, BG and GM detection are useful tests for the diagnosis of IA in high-risk hematological patients, but a combination of the two tests was very useful to identify false-positive reactions by each test.

Adolescent↗

[Co-existence of Kala-azar and salmonellosis in a 16 month-old baby? Or a false positive widal reaction in Kala-azar?].

A 16 month-old boy was referred to our hospital with a prolonged fever, hepatosplenomegaly, anemia and leucopenia. Visceral leishmaniasis was the first suspected diagnosis but the bone-marrow aspiration material revealed no Leishmania amastigotes. As the group agglutination test for Salmonella typhi-O was positive at 1/320 dilution, a diagnosis of salmonellosis was made and meropenem therapy was started. Although the fever decreased on the fourth day of therapy, the expected clinical improvement did not occur and there was a recurrence of the fever. At that point even though his parents were told about the seriousness of his condition, they had the patient charged from the hospital. About one week later, the patient was hospitalized again with the same complaints. His organomegaly had increased and his pancytopenia was more severe at this time. The re-assessment of the patient raised again the suspicion of kala azar. IFAT for Leishmania infection demonstrated a high level of positivity (1/1024) and a second bone-marrow aspiration material revealed Leishmania amastigotes. After the patient was started on meglumine antimonate therapy, his clinical and laboratory findings improved. The case has been evaluated according to two different points of view based on the co-existence of salmonellosis and kala azar or a possible false positive Widal reaction in kala-azar.

English Abstract↗

Protein A-antibody mediated hemagglutination assay for serodiagnosis of amebic liver abscess.

A successful modification of the indirect hemagglutination test to demonstrate antibodies for serodiagnosis of amebic liver abscess has been described in the present study. In the modified test, the protein A-IMA, Staphylococcus aureus (Cowan's strain I) bearing protein A (SAPA) cells were used to enhance hemagglutination of sensitized red cells. Use of SAPA cells markedly enhanced sensitivity of the test and greatly increased the titers obtained with most of the sera. At a diagnostic antibody titer of 1:128 and above, the protein A-IHA could detect 72 (100%) of amebic liver abscess (ALA) cases. Amongst the controls, no false positive reaction was observed in non-amebic liver disease controls. However 1(2%) of sera demonstrated false positive reactions from healthy controls. The protein A-IHA was highly sensitive when compared with that of the indirect hemagglutination (IHA) for serodiagnosis of amebic liver abscess. The novel immunoassay is simple, inexpensive and requires little technical skill. It has the potential for wide application in serodiagnosis of amebic liver abscess.

Animals↗

Accumulation of 99mTc-glutathione in head and neck tumors.

Glutathione labelled with 99mTc was used to study blood clearance and normal distribution in 3 healthy volunteers and in 10 patients with biopsy-proven tumors in the head and neck region. Static scintigrams were obtained at 1, 3, 6, and 24 h. ROIs over tumors and normal soft tissues were compared to obtain T/N ratios. In normal subjects blood clearance reached a plateau at 6 h; no radio-activity accumulation in the head and neck region was observed. Only the cardiac blood pool, the liver, the kidneys and the urinary bladder were evident. Excretion was via the kidneys. Malignant tumors and metastases were well visualized in 7 patients (true-positive), starting at 1 h. The mean T/N ratio was 2.69 +/- 0.77. The best images were obtained at 3-6 h. 1 false-positive (granulamatous reaction), 1 false-negative (malignant epithelial tumor in the radix of tongue) and 1 true-negative (angiofibroma) results were obtained. 99mTc-GSH is a potential radiopharmaceutical for the scintigraphic visualization of head and neck tumors. Further clinical studies are warranted to show its sensitivity and accuracy.

Adult↗

Discrepancies between two procedures for ds-DNA antibody detection: Farr test and indirect immunofluorescence on Crithidia luciliae.

Of the 54,911 sera routinely tested for anti-double stranded-DNA antibodies (Ab), 2,297 gave a positive reaction with both indirect immunofluorescence (IIF) on Crithidia luciliae (CL) and the Farr test, or with only one of the two tests. Of the sera giving positive reactions, only 1,499 (65.3% of the positive sera) were positive with both reactions. Among the remaining 798 sera (34.7% of positive sera), 48.25% gave a positive reaction with the Farr test and 51.75% with the IIF reaction. Of the discrepant Farr test(+), IIF-CL(-) sera, slightly fewer than half corresponded to a false positive reaction of the Farr test due to the presence in the serum of proteins other than Ab which were able to bind to the labelled ds-DNA to form a complex precipitable by 50% saturated ammonium sulfate solution. Slightly more than half of the other Farr test(+) IIF-C'(-) sera corresponded to a defect in the IIF-CL reaction. Among the discrepant Farr test(-) IIF-CL(+) sera, 1/3 corresponded to false positive reactions IIF-CL and 2/3 of the remaining sera contained weakly avid anti-ds-DNA antibodies undetectable using the Farr test.

Animals↗

Effect of distance between sites and region of the body on results of skin prick tests.

BACKGROUND: Two variables in skin prick testing were examined: the effect of a positive reaction to histamine or allergen on adjacent negative test sites and the relative size of reactions to allergen and histamine on the back and forearm. METHODS: Fifty-two subjects were tested on the back and arm with an allergen extract to which they reacted with a wheal of 8 mm or greater in diameter and with 50% glycerin at sites ranging from 2 to 7.5 cm distant from the allergen test site. A similar study was performed in 50 subjects by using 1.8 mg/ml histamine base to produce the positive reaction. The data were analyzed for the effect of a positive reaction on an adjacent negative control site and also for the relative size of positive reactions on the back and forearm. Data from a second study were also examined for the difference in reactivity on the back and arm. In this study 77 subjects underwent skin prick testing (in duplicate) with seven allergen extracts, 50% glycerin, and 1.8 mg/ml histamine base on the forearm and back. RESULTS: Positive reactions at the 50% glycerin sites were observed at a rate of 0.52% when defined as wheals of 3 mm in diameter or greater and at a rate of 1.11% when defined as flares of 10 mm in diameter or greater. There was no increase in the rate of false-positive reactions over the range of 2 to 5 cm distance from the sites of positive reactions to allergen or histamine. Fewer reactions were present at the 50% glycerin sites, 7.5 cm from the positive sites. The diameters of the reactions to allergen in the primary study were significantly (p < 0.001) smaller on the forearms than on the back (27% less for the wheal and 14% less for the flare). The mean diameter of the wheals induced by histamine did not vary between back and arm (p = 0.3); however, the mean diameter of the flares on the forearm with histamine were 8% smaller than those on the back (p = 0.003). In the second study the reactions to cat extract (n = 59) were significantly smaller on the forearm than on the back (mean wheal diameter 16% less, mean flare diameter 14% less, p < 0.001 for both). The reactions to histamine (n = 76) in this study were also significantly smaller on the forearm (mean wheal diameter 6% less, p = 0.027, and mean flare diameter 14% less, p < 0.001). CONCLUSION: If a relatively nontraumatic technique for skin testing is used, adjacent positive test reaction sites of moderate size, which are 2 cm or more from the site of the prick test, are unlikely to cause false-positive reactions on either the back or forearm. Skin test reactivity is less notable on the forearm than on the back for both histamine and allergen. This effect was more pronounced for allergen tests (16% to 27% decrease in mean wheal diameter) than for histamine tests.

Adolescent↗

An enzyme labelled nuclear antigen immunoassay for detection of cytomegalovirus IgM antibodies in human serum: specific and non-specific reactions.

A mu-capture enzyme linked immunosorbent assay was developed for detection of IgM antibody to cytomegalovirus (CMV). Virus-specific IgM was detected using horseradish peroxidase labelled nuclear CMV antigen (CMV-ELA). False-positive reactions caused by Paul-Bunnell-Davidsohn (PBD) positive sera and antinuclear antibody (ANA) positive sera were identified in a combination assay employing enzyme labelled nuclear control antigen (CO-ELA) in parallel to the CMV-ELA. Four of five PBD positive and 30 of 31 ANA positive sera reactive with the CMV-ELA were identified as false positive reactions in the combined ELA-assay. The reactivity in PBD-positive sera could not be explained by antigenic cross reactivity between CMV and Epstein-Barr virus, and the results further suggested that different cell specified components of the CMV-ELA were responsible for the reactivity of PBD-positive as compared to ANA-positive sera. One of 314 healthy blood donors, 12 of 12 patients with primary CMV infection, and 11 of 15 patients with secondary CMV infection had detectable CMV IgM antibodies. Comparison of different CMV-ELAs revealed that pronounced differences in specificity as well as sensitivity may exist.

Antibodies, Antinuclear↗

Syphilis serology and HIV infection in Harare, Zimbabwe.

OBJECTIVE: To determine the reliability of serological tests in detecting syphilis in a factory worker cohort and examine the impact of concurrent HIV infection on serological tests for syphilis. METHOD: Reactions to non-treponemal and treponemal antigens were tested using sera from a cohort of 3401 factory workers in Harare, Zimbabwe. The participants consented to regular testing for syphilis, by VDRL, and HIV using two ELISAs. All sera from men who were VDRL positive, and a random sample of VDRL negative sera, were tested by RPR, TPHA, and where appropriate FTA-Abs. From the results, men were defined as having no syphilis, active syphilis, incident syphilis, historic syphilis, or giving biological false positive reactions. RESULTS: 709 sera were examined from 580 men. There were 78 cases of active syphilis in the cohort, giving a prevalence of 2.3%, and the seroincidence was 0.25 per 100 person years of follow up. The prevalence of HIV in the cohort was 19.8%. There was a strong association between syphilis, whether active, incident or historic, and HIV seropositivity. With both HIV positive and negative sera the negative predictive values of VDRL and RPR were > 99.9% while the positive predictive value for VDRL (30%) was lower than for RPR (39%). Biological false positive reactions were detected in 0.5% of the cohort, with in most cases a transient rise in VDRL titres up to < 1/16. Higher false positive titres occurred in five men, each of whom was HIV positive. CONCLUSIONS: The VDRL is reliable in detecting possible cases of syphilis even in a community with a high prevalence of heterosexually transmitted HIV. There is need, however, for confirmatory tests. The prevalence of syphilis in this cohort is very low in comparison with other countries in southern Africa, but is consistent with recent data from Harare. Despite a strong association between syphilis and HIV it was clear that syphilis could not be counted as a major factor fueling the HIV epidemic in Zimbabwe.

Cohort Studies↗

Leishmanin skin sensitivity in the inland delta of the Niger.

Leishmanin skin tests were applied to 266 subjects in the city of Mopti which is located on several islands in the center of the inland delta of the Niger in the Republic of Mali. The antigen employed contains 6-10 million killed leptomonads per ml in a sterile suspension preserved with 1:10,000 thiomersal and unlike the phenol suspended antigens, does not give false positive tests. Of the 249 subjects who completed the study 12 (4.8%) had positive reactions, the lowest positive reaction rate yet observed in Mali. No false positive reactions occurred among 139 subjects tested with a control solution of 1:10,000 thiomersal. The absence of known cases of cutaneous leishmaniasis in Mopti's permanent population and the absence of suitable vectors within the city would indicate that positive reactors reflect previous L. tropica infections acquired in the surrounding rural areas where the disease is known to exist or else previous infection with non-pathogenic mammalian or reptilian species of Leishmania.

Adolescent↗

Investigation of apparent false-positive urine latex particle agglutination tests for the detection of group B streptococcus antigen.

In our nursery, we identified neonates with positive urine latex particle agglutination (LPA) tests for group B streptococcus (GBS) antigen who did not have corroborating cultural evidence of infection. To investigate the mechanisms underlying these apparent false-positive reactions, we examined the urine LPA test in an unselected population of neonates suspected of sepsis. Urine specimens for LPA testing and culture and simultaneous perirectal cultures were obtained from 134 neonates with suspected sepsis. Six infants had positive blood cultures for GBS; four of the six were positive by LPA testing. An additional 20 infants had positive LPA tests but negative blood cultures; of these, 13 had mothers who received antibiotic treatment prior to delivery. Two infants with positive LPA results and negative blood cultures had bacteria isolated from urine cultures obtained in a nonsterile fashion (GBS, Escherichia coli). GBS was not isolated from perirectal swabs of infants with positive LPA tests and negative blood cultures. In conclusion, (i) a high proportion of neonates evaluated for sepsis gave positive LPA tests and negative blood cultures, (ii) local contamination of the perirectal skin or urinary tract with GBS was an unlikely source of false-positive LPA reactions, and (iii) maternal antibiotic pretreatment during labor may represent an important cause of apparent false-positive LPA reactions.

Anti-Bacterial Agents↗

Evaluation of a 23S rDNA polymerase chain reaction assay for identification of Serpulina intermedia, and strain typing using pulsed-field gel electrophoresis.

A polymerase chain reaction assay, amplifying a 1027 base pair portion of the 23S rDNA gene, was evaluated for identification of the intestinal spirochaete Serpulina intermedia. A total of 34 strains of S. intermedia isolated from pigs and chickens and 195 strains of other related species were tested. The optimised assay correctly identified all the S. intermedia strains, but generated 11 false positive reactions, giving a test sensitivity of 100% and a test specificity of 94.3%. The false positive reactions were generated from strains of four different species of intestinal spirochaetes, and the product was of the original predicted size. This suggests that the primer sites selected on the 23S rRNA gene were not completely specific for S. intermedia. Pulsed-field gel electrophoresis was then developed to investigate diversity amongst the S. intermedia strains. All strains tested had distinct DNA banding patterns using Mlu1, although three isolates from chickens on the same farm appeared closely related. The collection exhibited considerable genetic diversity, and strains from pigs and chickens were distributed in clusters throughout the dendrogram produced. The most closely related porcine and avian strains shared only 62% similarity.

Animals↗

Development of a highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses enzyme-labeled antigen.

An enzyme immunoassay (EIA) for serum immunoglobulin M (IgM) antibodies to rubella virus based on enzyme labeling of viral antigen was developed. The sensitivity of the EIA for the detection of recent rubella virus infection was evaluated by using 115 rubella-IgM-antibody-positive serum specimens, which were confirmed as positive by Rubazyme M (Abbott Diagnostics). In addition, 12 individuals, 2 of whom were exposed to rubella through vaccination and 10 of whom were exposed through natural infection, were studied, and the results were compared with those obtained by indirect EIA (Rubelisa M; Electro-Nucleonics, Inc.) and immunoblotting. The sensitivity of the newly developed EIA with sera from these individuals was 100%. Serum specimens from two patients indicated that the IgM antibodies were detected by the newly developed EIA at the same time as IgM antibodies were detected by immunoblotting and before positive reactions were detected by an indirect EIA. The reference population consisted of 564 healthy blood donors and hospitalized patients (150 serum specimens). In addition, 145 serum specimens commonly giving false-positive reactions in conventional rubella IgM EIAs were studied. With these specimens, no false-positive reactions were observed. Positive IgM responses, which could not be confirmed by immunoblotting, were observed in two samples from the reference population. However, these two samples were rubella IgG positive. The overall specificity of the EIA was 99.8%.

Antibodies, Viral↗

Cross-reactions in immunodiagnosis of patients infected with Schistosoma, Fasciola and Heterophyes using ELISA.

Cross-reactions in immunodiagnosis using schistosomal soluble egg antigen (SEA) and Fasciola adult worm antigen in cases with fascioliasis, heterophyiasis and schistosomiasis were studied by ELISA techniques. False positive reactions occurred in 10% of cases with fascioliasis and heterophyiasis respectively when using schistosomal SEA. Using Fasciola crude antigen, false positive reaction occurred in 25% of cases with schistosomiasis and 35% with heterophyiasis. The specificity of SEA in heterologous cases was 87% while that of Fasciola antigen was 70%. This difference was found to be statistically significant.

Animals↗

[Comparison of 3 techniques in the serological diagnosis of human hydatidosis].

Three serological techniques for hydatid disease were compared using 60 human serum samples obtained from 29 patients with hydatidosis previously demonstrated by surgery, 15 apparently healthy individuals and 16 persons with different pathologies. The results obtained with these 3 techniques showed some differences that could associate with the anatomical location of the parasite and the biological stage of the hydatid cysts. The global sensitivity of the techniques was 96.6% for ELISA, 86.2% for IHAT and 79.3 for DD5. False positive reactions were observed with IHAT and ELISA in sera from patients with infection by Taenia saginata. ELISA also showed cross reactions in the sera of four patients with intestinal infection by Ascaris lumbricoides. No false positive reactions were observed with DD5 test.

Adolescent↗

Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev.1-vaccinated sheep.

The problem of differentiating sheep infected with Brucella melitensis from those vaccinated or exposed to cross-reaching organisms has not been resolved by conventional serological tests or through the use of the smooth lipopolysaccharide in primary binding assays. We therefore analyzed sera from ewes experimentally infected with B. melitensis H38, from ewes naturally infected with B. melitensis, and from B. melitensis Rev.1-vaccinated ewes by enzyme-linked immunosorbent assay with three antigenic fractions: O polysaccharide, a cytosoluble protein extract (CPE) from the rough strain B. melitensis B115, and a partially purified cytosoluble protein of 28 kDa (CP28) from the CPE. Immunoglobulin G anti-O polysaccharide and anti-CPE responses were detected in all groups of animals tested (Rev.1 vaccinated and B. melitensis infected). However, false-positive reactions with CPE occurred with sera from Brucella-free ewes. The use of partially purified CP28 abolished these false-positive reactions. Furthermore, no immunoglobulin G antibodies against CP28 were detected in sera from vaccinated ewes, whereas 80% (8 of 10) of ewes experimentally infected with B. melitensis H38 and 89% (25 of 28) of naturally infected ewes showed various degrees of anti-CP28 reactivity (absorbance values of between 0.5 and 2.5). The results obtained with CP28 showed the potential usefulness of this antigen to permit the detection of B. melitensis-infected ewes and their differentiation from B. melitensis Rev.1-vaccinated ones.

Animals↗

HBsAg immune complex glomerulonephritis.

Out of 97 patients with various forms of glomerulonephritis 10 were found to be HBsAg seropositive. None of these showed HBsAg immunodeposits within the kidney. Direct immunofluorescent kidney staining for HBsAg was observed in 4 out of 87 HBsAg seronegative patients. The HBsAg staining in kidneys was a false positive reaction due to binding of the Fc portion of the fluoresceinated IgG molecules to the IgM RF tissue deposits. The false positive reaction for HBsAg is not revealed by the usual specificity controls for fluorescence staining. The role of HBsAg in glomerulonephritis remains unproven.

Antigen-Antibody Complex↗

Specificity of the Crithidia luciliae method for detecting anti-DNA antibodies. Effect of absorption for lipoproteins.

Using the immunofluorescent (IF) assay with Crithidia luciliae smears, anti-native (n) DNA antibodies were detected in the sera of 12 of 20 systemic lupus erythematosus (SLE) patients, in 1 of 6 mixed connective tissue disease cases, in 2 of 38 patients with systemic sclerosis but in none of the sera from 96 normal subjects. All anti-nDNA antibodies were associated with antinuclear antibodies (ANA). However, occasionally sera were encountered in routine screening which appear to be positive for anti-DNA antibodies but negative for ANA. Studies of such sera indicate that this is a nonspecific reaction which can be abolished by treating sera with dextran sulfate or heparin. Treatment of SLE sera with these agents had no effect on their anti-nDNA antibody activity. Absorption of sera with Aerosil eliminated the false positive reactions with C. luciliae; however, this treatment also removed immunoglobulins, ANA and anti-nDNA antibodies. Evidence is reviewed which points to a role of complexes of low density lipoprotein and IgG in the nonspecific binding reactions with C. luciliae which is seen as false positive reactions for anti-nDNA antibodies.

Adsorption↗

Effects of urinary metabolites of etodolac on diagnostic tests of bilirubin in urine.

Etodolac (CAS 41340-25-4) is a non-steroidal anti-inflammatory drug used clinically. The bilirubin levels in urine samples from patients treated with etodolac were determined with diagnostic tests (diazo and oxidized methods). The urine samples gave a positive reaction with a diazo method, but not with an oxidized method. The serum concentrations of bilirubin in the patients were also in the normal range. These results indicate that the positive reaction of the urine samples from patients treated with etodolac is false and caused by urinary metabolites of etodolac. To identify the false positive substance in the urine samples, the urinary metabolites of etodolac were extracted with ethyl acetate. The extract was injected into a high performance liquid chromatography (HPLC) column and the eluate was mixed with the diazo reagent. Three positive fractions were found. The retention times of the two metabolites in HPLC matched with those of the 6- and 7-hydroxylated metabolites of etodolac. In addition, authentic compounds of the 6- and 7-hydroxylated metabolites gave a positive reaction in the diazo methods. These results indicate that the 6- and 7-hydroxylated metabolites are mainly responsible for the false positive reactions of the urine sample from patients treated with etodolac.

Aged↗