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PubMed2000

Analytical performance and clinical utility of a nucleic acid sequence-based amplification assay for detection of cytomegalovirus infection.

A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection.

Antigens, Viral↗
PubMed2000

Serodiagnosis of Bartonella bacilliformis infection by indirect fluorescence antibody assay: test development and application to a population in an area of bartonellosis endemicity.

Bartonella bacilliformis causes bartonellosis, a potentially life-threatening emerging infectious disease seen in the Andes Mountains of South America. There are no generally accepted serologic tests to confirm the disease. We developed an indirect fluorescence antibody (IFA) test for the detection of antibodies to B. bacilliformis and then tested its performance as an aid in the diagnosis of acute bartonellosis. The IFA is 82% sensitive in detecting B. bacilliformis antibodies in acute-phase blood samples of laboratory-confirmed bartonellosis patients. When used to examine convalescent-phase sera, the IFA is positive in 93% of bartonellosis cases. The positive predictive value of the test is 89% in an area of Peru where B. bacilliformis is endemic and where the point prevalence of infection is 45%.

Antibodies, Bacterial↗
PubMed2001

Validation of a commercially available monoclonal antibody-based competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to Neospora caninum in cattle.

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody to Neospora caninum, a major cause of bovine abortion. Modifications to the cELISA included capturing native N. caninum antigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a "gold standard" set of 184 cow sera defined by fetal histopathology and N. caninum immunohistochemistry and by maternal N. caninum indirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknown N. caninum status. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknown N. caninum status revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modified N. caninum cELISA provided a simple, rapid, and versatile method to accurately identify N. caninum infection status in cattle using a single cutoff value.

Abortion, Veterinary↗
PubMed2002

Molecular identification and typing of enteroviruses isolated from clinical specimens.

Enterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5' noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.

Capsid Proteins↗
PubMed2002

Seroprevalence of Babesia infections in humans exposed to ticks in midwestern Germany.

Babesiosis is considered to be an emerging tick-borne disease in humans worldwide. However, most studies on the epidemiology of human babesiosis to date have been carried out in North America, and there is little knowledge on the prevalence of infection and frequency of disease in other areas. The aim of this study was to investigate the prevalence of Babesia infections in a human population in Germany. A total of 467 sera collected between May and October 1999 from individuals living in the Rhein-Main area were tested for the presence of immunoglobulin G (IgG) and IgM antibodies to antigens of Babesia microti and Babesia divergens by indirect fluorescent-antibody (IFA) tests. These sera were derived from 84 Lyme borreliosis patients suffering from erythema migrans, 60 asymptomatic individuals with positive borreliosis serology, and 81 individuals with a history of tick bite. Cutoff values for discrimination between seronegative and seropositive results in the IFA tests were determined using sera from 120 healthy blood donors and 122 patients suffering from conditions other than tick-borne diseases (malaria, n = 40; toxoplasmosis, n = 22; syphilis, n = 20; Epstein-Barr virus infection, n = 20; and presence of antinuclear antibodies, n = 20). The overall specificities of the IFA tests for B. microti and B. divergens were estimated to be >or=97.5%. Positive IgG reactivity against B. microti antigen (titer, >or=1:64) or B. divergens antigen (titer, >or=1:128) was detected significantly more often (P < 0.05) in the group of patients exposed to ticks (26 of 225 individuals; 11.5%) than in the group of healthy blood donors (2 of 120 individuals; 1.7%). IgG antibody titers of >or=1:256 against at least one of the babesial antigens were found significantly more often (P < 0.05) in patients exposed to ticks (9 of 225) than in the control groups (1 of 242). In the human population investigated here, the overall seroprevalences for B. microti and B. divergens were 5.4% (25 of 467) and 3.6% (17 of 467), respectively. The results obtained here provide evidence for concurrent infections with Borrelia burgdorferi and Babesia species in humans exposed to ticks in midwestern Germany. They also suggest that infections with Babesia species in the German human population are more frequent than believed previously and should be considered in the differential diagnosis of febrile illness occurring after exposure to ticks or blood transfusions, in particular in immunocompromised patients.

Adult↗
PubMed2002

Seroprevalence of antibodies that react with Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis, in different populations in Westchester County, New York.

We determined the frequencies of antibodies to Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), in different groups of adults and children from Westchester County, New York. The groups included 159 adult blood donors and 215 children who were seronegative for Borrelia burgdorferi antibodies, 118 adult patients and 57 children who were seropositive for B. burgdorferi antibodies, and 42 adult patients with culture-confirmed erythema migrans. Eighteen (11.3%) of the blood donors and 11 (5.1%) of the B. burgdorferi-seronegative children were found to have A. phagocytophila antibodies by indirect immunofluorescent-antibody assay (IFA). Nine of 42 (21.4%) patients with culture-confirmed erythema migrans tested at the baseline visit, 42 of 118 (35.6%) adults, and 3 of 57 (5.3%) children whose sera were reactive for B. burgdorferi antibodies also tested positive for A. phagocytophila antibodies. The geometric mean titer ranged from 219 to 315 for all groups, and the differences in titers among the groups were not statistically significant. Only one-third of the healthy blood donors reactive by IFA were confirmed to be positive by immunoblotting. We conclude that a significant proportion of adults and children without clinical evidence of HGE will test positive for A. phagocytophila antibodies when the conventional cutoff titer of 80 is used in the IFA. This information must be considered in interpretation of test results.

Adolescent↗
PubMed2005

Value of serological testing for diagnosis of legionellosis in outbreak patients.

Serum antibody detection tests and a urine antigen detection technique were compared in samples from 116 patients epidemiologically characterized as belonging to a legionellosis outbreak. Sera were tested by enzyme-linked immunosorbent assays (ELISAs) for immunoglobulin M (IgM) and IgG plus IgM and by immunofluorescent assays (IFAs) for IgG, IgM, IgA, and polyimmunoglobulin using commercial kits (Vircell); concentrated urines were tested with the Binax NOW Legionella test. ELISA for IgM, ELISA for IgG plus IgM, antigenuria detection, and IFA for IgM were able to diagnose 72.3%, 60.5%, 53.3%, and 51.4%, respectively, of patients. Antigenuria was present in 53.8% of first samples, ELISA detected IgM in 29.7%, ELISA detected IgG plus IgM in 7.9%, and IFA detected IgM in 3.9%. Ten antigenuria-negative first samples tested serologically positive, 9 of them to IgM by ELISA. Despite the single source of the samples included in the study, detection of IgM using a sensitive technique such as ELISA seems to be a suitable complement to antigenuria detection for the diagnosis of legionellosis.

Adult↗
PubMed1977

Comparison of rabies humoral antibody titers in rabbits and humans by indirect radioimmunoassay, rapid-fluorescent-focus-inhibition technique, and indirect fluorescent-antibody assay.

Rabies humoral antibodies were induced in eight New Zealand rabbits by a single intramuscular injection of inactivated suckling mouse brain rabies vaccine. The primary response to immunization was measured in blood samples taken at selected intervals for 6 months. The anamnestic response was measured in blood samples obtained 2 weeks after the rabbits received a booster immunization. The humoral antibody concentrations were measured by the rapid-fluorescent-focus-inhibition technique (RFFIT), indirect fluorescent-antibody assay (IFA), and indirect radioimmunoassay (RIA). The maximal neutralizing antibody titers as measured by RFFIT were attained by the 4th week and persisted into the 24th week. After booster immunization the antibody response was almost 10-fold higher than the highest level attained in the primary response. The antibody levels as measured by IFA and RIA were similar, but the titers as measured by either procedure were almost 10-fold lower than those determined by RFFIT. After booster immunizations the antibody levels, as measured by IFA and RIA, were three- and sixfold higher, respectively, than the maximal levels attained in the primary response. Twenty-two human serum specimens were tested by the same serological procedures, with disparate results. Both RIA and RFFIT effectively differentiated antirabies-positive sera from antirabies-negative sera.

Animals↗
PubMed1988

Fluorescein angiography of anterior uveal melanocytic tumours.

A retrospective analysis of 32 cases of anterior uveal melanocytic tumours included 14 cases in which the tumours could be categorised histologically as malignant, of intermediate cytology with equivocal features of malignancy, or as benign progressive naevi. An additional eight cases without a histological diagnosis were included as benign naevi, because there had been no change in their clinical characteristics after a minimum follow-up of four years. Clinical features and iris fluorescein angiographic (IFA) findings were analysed with respect to these groups in an attempt to identify features predictive of malignant or locally invasive behaviour. Very small tumours were more likely to be benign (p = 0.029). Glaucoma and episcleral vascular dilatation occurred in ciliary body tumours only. Tumours involving the anterior chamber angle proved more likely to be malignant (p = 0.019). IFA showed a disorganised vasculature and gross late leakage in 4/7 (56%) malignant melanomas as well as in 2/6 (33%) tumours with intermediate cytology. No benign tumours showed these features. Four out of nine (44%) benign tumours, but no tumours with malignant or intermediate cytology, showed complete masking of fluorescence. Early leakage of dye from tumour vessels and a geometric tumour vasculature were not specific features of any tumour category. IFA correlated with the cytology and behaviour of anterior uveal melanocytic tumours in 11/22 (50%) of our cases.

Adult↗
PubMed1989

Comparison of immunofluorescence and enzyme linked immunosorbent assays for diagnosing Lyme disease.

A new enzyme linked immunosorbent assay (ELISA) method was assessed in parallel with an indirect immunofluorescence assay (IFA) for the serological testing of specimens submitted for detection of Lyme disease. Wells of an ELISA microtitre plate were coated with sonicated whole cells of Borrelia burgdorferi in carbonate buffer. After overnight incubation at 4 degrees C the plates were washed three times, then incubated at 37 degrees C for an hour sequentially with a blocking solution, diluted test serum, anti-human IgG horseradish peroxidase conjugate, and finally for half an hour at room temperature with o-phenylenediamine substrate before being read at 492 nm. Absorbance results were converted into arbitrary ELISA units. Of a total of 1760 sera, 146 (8.3%) were positive: of these 92 (63%) were positive by both methods, five (3.4%) by IFA alone, and 49 (33.6%) by ELISA alone. The ELISA was better suited for testing large numbers of specimens and easier to interpret than IFA.

Antibodies, Bacterial↗
PubMed1998

In situ localisation of Yersinia enterocolitica by catalysed reported deposition signal amplification.

AIM: The sensitive detection of pathogenic Yersinia enterocolitica in paraffin embedded tissue sections by in situ hybridisation (ISH). METHODS: Y enterocolitica infected cell lines, rat spleens, and patient biopsy specimens were used to compare conventional ISH, immune fluorescence assay (IFA) detection, and catalysed reporter deposition (CARD) signal amplification ISH. RESULTS: CARD-ISH was shown to be more sensitive then conventional ISH and had a comparable sensitivity to IFA. In contrast to IFA, CARD-ISH preserved good tissue morphology. CONCLUSIONS: CARD-ISH appeared to be a fast and sensitive ISH method for detecting Y enterocolitica in routinely processed tissue sections. Application of this method allows the combination of routine detection and cellular localisation of the pathogen within the infected tissue.

Animals↗
PubMed1977

Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain.

Use of the indirect fluorescent antibody (IFA) tests is described to detect antibodies to Theileria mutans and Babesia major in the sera of infected cattle. When antisera against T mutans and B major were tested against homologous antigens high antibody titres were recorded: when they were tested against each other or against Babesia divergens antigen insignificant titres (1/40 or less) were recorded. Thus the test was found to be species specific. Animals recovered from T mutans and B major infections retained significant levels of IFA titres for 22 and 11 months respectively. It is suggested that the IFA test could be used for field survey of the piroplasms of cattle in Britain.

Animals↗
PubMed1990

The immune response in a cat-related outbreak of Q fever as measured by the indirect immunofluorescence test and the enzyme-linked immunosorbent assay.

The isotypic immune response of 16 individuals who developed Q fever pneumonia following exposure to an infected parturient cat was studied. The enzyme-linked immunosorbent (ELISA) test was used to detect IgM, IgA, and IgG antibodies to phase I and phase II Coxiella burnetii whole-cell antigens and to the phase I lipopolysaccharide. The indirect immunofluorescent antibody (IFA) test was also used to detect antibodies to phase I and phase II whole cells. None of the 16 subjects developed antibodies to the phase I lipopolysaccharide. The ELISA was more sensitive than the IFA test. IgM antibodies to phase II antigen were detectable by ELISA in 80% of the subjects at the time of onset of symptoms and were still present in 7 of the 8 tested at 32 weeks following the onset of symptoms. In all instances (ELISA: IgG, IgM; IFA: IgG, IgM) phase II antibodies developed earlier and reached higher levels than did phase I antibodies. The absence of antibodies to phase I lipopolysaccharide in acute Q fever combined with our unpublished findings of antibodies to phase I lipopolysaccharide in chronic Q fever suggests that this test may be used to distinguish acute from chronic Q fever.

Adult↗
PubMed1997

A rapid viability assay for Cryptosporidium oocysts and Giardia cysts for use in conjunction with indirect fluorescent antibody detection.

There is an urgent need to develop rapid methods to determine the viability of Cryptosporidium oocysts and Giardia cysts in environmental samples, especially water. The inclusion of the vital dye propidium iodide (PI) by oocysts and cysts has been previously shown to correlate well with nonviability. The ability of nonviable oocysts and cysts to include PI has been employed to develop a rapid viability determination method that could be used in conjunction with the current indirect fluorescent antibody (IFA) method for detecting oocysts and cysts. The efficacy of this PI-IFA method to detect and determine the viability status of oocysts/cysts has been tested using oocyst samples inactivated by three different approaches. The ability to incorporate PI staining with IFA detection provides the advantage of both detection and viability determination at the same time, using the same sample.

Animals↗
PubMed2001

Growth hormone pulsatility profile characteristics following acute heavy resistance exercise.

This investigation examined the hypothesis that acute heavy resistance exercise (AHRE) would increase overnight concentrations of circulating human growth hormone (hGH). Ten men (22 +/- 1 yr, 177 +/- 2 cm, 79 +/- 3 kg, 11 +/- 1% body fat) underwent two overnight blood draws sampled every 10 min from 1700 to 0600: a control and an AHRE condition. The AHRE was conducted from 1500 to 1700 and was a high-volume, multiset exercise bout. Three different immunoassays measured hGH concentrations: the Nichols immunoradiometric assay (Nichols IRMA), National Institute of Diabetes and Digestive and Kidney Diseases radioimmunoassay (NIDDK RIA), and the Diagnostic Systems Laboratory immunofunctional assay (DSL IFA). The Pulsar peak detection system was used to evaluate the pulsatility profile characteristics of hGH. Maximum hGH was lower in the exercise (10.7 microg/l) vs. the control (15.4 microg/l) condition. Mean pulse amplitude was lower in the exercise vs. control condition when measured by the Nichols IRMA and the DSL IFA. A differential pattern of release was also observed after exercise in which hGH was lower in the first half of sleep but higher in the second half. We conclude that AHRE does influence the temporal pattern of overnight hGH pulsatility. Additionally, because of the unique molecular basis of the DSL IFA, this influence does have biological relevance because functionally intact molecules are affected.

Adult↗
PubMed2002

Review of an inactivated vaccine against hantaviruses.

OBJECTIVE: Hantaviruses cause haemorrhagic fever with renal syndrome and result in severe morbidity and mortality in humans. Safe and effective vaccines are needed to reduce the incidence of human illness. In this study, the immune response to an inactivated hantavirus vaccine was measured in 64 human volunteers for Hantavax and 10 human volunteers for a Hantaan-Puumala virus combination vaccine at high risk of infection by virtue of their residence and occupation. METHODS: A serum sample was obtained from each volunteer before the initial vaccination (day 0), 30 days after each inoculation and 1 year after the initial dose. All sera were kept at -20 degrees until tested. IgG-specific antibody titres were tested by ELISA and immunofluorescence assay (IFA). Neutralizing antibody titres were determined by a plaque reduction neutralizing test. RESULTS: Thirty days after vaccination, 79 and 62% of the subjects had developed a significant hantavirus antibody titre as measured by IFA and ELISA, respectively. Seroconversion rates increased to 97% 1 month after the booster dose. Neutralizing antibody titres paralleled this trend, with 13% of vaccine recipients producing neutralizing antibody 1 month after the first dose and 75% of vaccine recipients responding 1 month after boosting. Antibody titres had declined by 1 year, however, with only 37 and 43% of sera found to be positive by IFA and ELISA, respectively. Re-vaccination at this time produced a vigorous anamnestic response, with 94 and 100% of vaccine recipients yielding positive antibody titres. Only 50% of the sampled population, however, produced neutralizing antibodies following the booster dose 1 year later. CONCLUSIONS: The vaccine was well tolerated and there were no apparent differences in the responses in human subjects. However, further improvement of this vaccine is necessary in order to induce a longer-lasting humoral immune response.

Adult↗
PubMedDate not supplied

Cross-reaction of sera from patients with various infectious diseases with Leishmania infantum.

OBJECTIVE: To compare and evaluate the application of indirect fluorescent antibody (IFA) and counterimmunoelectrophoresis (CIEP) for laboratory identification of visceral leishmaniasis. MATERIALS AND METHODS: Serum samples from patients with malaria (Plasmodium vivax, n = 86; Plasmodium falciparum, n = 38), brucellosis (n = 26), tuberculosis (n = 31) and typhoid fever (n = 35) were examined for the presence of antibody to Leishmaniainfantum antigen using IFA and CIEP tests. RESULTS: Using IFA, false-positive results were malaria (P. vivax 19.8%, P. falciparum 13.2%), tuberculosis (6.4%), brucellosis (3.8%), and typhoid fever (2.8%). Using CIEP, a lower percentage of false-positives was observed only among malaria patients (P. vivax 2.3%, P. falciparum 2.6%). Serum samples from patients with other infectious diseases were negative in the CIEP test. CONCLUSION: Based on the results of this study, the CIEP technique is recommended for immunodiagnosis of visceral leishmaniasis, especially in regions where malaria, brucellosis and tuberculosis are prevalent.

Communicable Diseases↗
PubMed1979

Farmer's lung disease in Vermont.

Several Vermont population groups were surveyed for the occurrence of antibodies to thermophilic actinomycetes. Antibodies to M. faeni and T. vulgaris were measured by the precipitin method in all subjects and, in 124 subjects, M. faeni antibodies were also measured by the indirect fluorescent antibody (IFA) technique. There was relatively good correlation between the two techniques (r 0.48, p less than 0.01). Hospital employees, blood donors and patients with chronic bronchitis were generally negative for precipitins to thermophilic actinomycetes. Of the 258 Vermont dairy farmers surveyed, 14 (5.4%) had precipitins to M. faeni, 3 (1.2%) had precipitins to T. vulgaris but only 1 farmer with antibodies to M. faeni had symptoms of possible farmer's lung disease (FLD). On the other hand, 10 (4.1%) precipitin-negative farmers had symptoms possibly consistent with FLD. The IFA test did not correlate any better with symptoms. 7 (5.6%) patients with pulmonary fibrosis had precipitins to M. faeni 5 of these were diagnosed as having FLD. 18 (14.4%) had precipitins to T. vulgaris and only 3 of these patients were felt to have hypersensitivity pneumonitis. Patients with pulmonary fibrosis and FLD hat IFA titers of greater than or equal to 1/128, but so did asymptomatic farmers.

Adolescent↗