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PubMed1995

Identification of enteroviruses by indirect immunofluorescence using monoclonal antibodies.

BACKGROUND: Serum neutralization (Nt) is used most often to type enterovirus isolates, but it is labor-intensive, expensive, and supplies of reference antisera for Nt are limited. Alternative methods of enterovirus typing are needed. OBJECTIVES: To investigate the use of indirect immunofluorescence (IFA) with commercially available monoclonal antibodies (MAbs) as an alternative to Nt for the identification of enteroviruses. STUDY DESIGN: Two MAb blends (one for coxsackie B viruses and one for echoviruses 4, 6, 9, 11, 30, and 34) and a coxsackie A9 MAb were used to screen 465 clinical isolates over a period of two years. Virus isolates which tested positive with one of the blends were typed with the individual MAbs of the respective blend. Individual MAbs for polioviruses 1, 2, and 3 acquired late in the study were used to screen 45 viral isolates. RESULTS: The antibodies identified 251/465 (54%) of the total number of isolates tested. IFA results for 451 of 465 viral isolates were in agreement with conventional identification methods. The sensitivity of the IFA screen using the MAb blends and coxsackie A9 MAb was 93% and the specificity was 99%. Thirteen discrepant isolates were negative by IFA, with twelve positive by Nt for echovirus 30 and one isolate positive by Nt for coxsackie A9. The remaining discrepant isolate was positive by IFA for both coxsackie A9 and coxsackie B5, but positive by Nt for coxsackie A9 only. CONCLUSIONS: IFA is highly specific for the identification of enteroviruses, but may not be sensitive enough to identify all strains within an enterovirus type. Procedures which utilize an IFA screen and confirm final results by Nt decrease turnaround time and reduce the number of cell culture tubes required for the identification of each enterovirus isolate.

Journal Article↗
PubMed2005

Immunization with gp120-depleted whole killed HIV immunogen and a second-generation CpG DNA elicits strong HIV-specific responses in mice.

HIV-1 Immunogen is a gp120-depleted whole killed virus vaccine candidate formulated with Incomplete Freund's Adjuvant (HIV-IFA). We evaluated in a mouse model the immunogenicity of HIV-IFA by itself and when combined with HYB2055, an immunomodulatory oligonucleotide consisting of a novel DNA structure and synthetic CpR immunostimulatory motif, as an adjuvant. C57/BL6 mice were immunized with HIV-IFA alone or combined with HYB2055. Mice treated with HYB2055 or with PBS were used as controls. Compared to HIV-IFA alone, immunization with HIV-IFA and HYB2055 combination elicited strong production of HIV- and p24-specific IFNgamma, RANTES, MIP 1alpha, and MIP 1beta, as well as high titers of HIV- and p24-specific antibodies. Inclusion of HYB2055 also reduced levels of IL-5 produced by HIV-IFA alone. HYB2055 enhances the immunogenicity of HIV-IFA and shifts responses towards a type 1 cytokine profile. The immune enhancing effects of HYB2055 adjuvant were dose-dependent. These findings warrant clinical evaluation of the HIV-1 immunogen/HYB2055 candidate as a therapeutic vaccine for HIV-1 infected patients.

AIDS Vaccines↗
PubMed2002

Antinuclear antibodies (ANA) screening by enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: analytical and clinical evaluation.

OBJECTIVES: Immunofluorescence assay (IFA) has been the standard method for antinuclear antibodies (ANA). To simplify and standardize the ANA test, generic ANA solid phase enzyme immunoassay has been promoted. The objective of the present work has been to study the relationship with IFA and the clinical usefulness of a generic EIA for ANA (COBAS Core HEp-2 ANA EIA, Roche Diagnostics). DESIGNS AND METHODS: We studied 74 healthy individuals, 119 patients with defined systemic autoimmune diseases, 26 patients with other autoimmune diseases, and 490 routine samples sent to laboratory for ANA analysis. RESULTS: Precision study showed intra-assay coefficient of variations (CVs) below 8% and inter-assay CVs below 10%. In relation to IFA, a 0.6 kappa index of agreement was obtained. COBAS-ANA concentrations increased according to IFA titer and greatest COBAS-ANA responses were obtained with pure or mixed homogeneous patterns and centromeric patterns. Analysis of COBAS-ANA response to particular antigenic specificities showed that SS-B, Scl-70 and U1sn-RNP specificities were saturating at high concentrations, whereas Jo-1, SS-A and nuclear and centromeric specificities exhibited lower responses. Elevated serum concentrations of IgG and IgM did not interfere COBAS-ANA, but high serum rheumatoid factor (RF) concentrations produced a decrease of ANA. For systemic lupus erythematosus (SLE) patients, the COBAS-ANA best efficiency was obtained with a cut-off of 0.9, with a sensitivity of 97% and a specificity of 88%, whereas the best IFA-ANA efficiency was obtained with a 1:80 dilution, giving a sensitivity of 90% and a specificity of 99%. There were no differences between areas under ROC curves for COBAS-ANA and IFA-ANA. For other systemic and nonsystemic autoimmune diseases sensitivity and specificity of COBAS-ANA were similar or higher than that of 1:160 IFA-ANA titer. CONCLUSION: Sensitivity and specificity of COBAS Core ANA-EIA for SLE and other systemic and nonsystemic autoimmune diseases, together with performance characteristics make it an adequate automated system for ANA screening.

Adult↗
PubMedDate not supplied

Oxygen radicals and nitric oxide production by turkey respiratory macrophages.

The influence of different induction protocols on the recovery of elicited turkey respiratory macrophages (RM), and on their oxygenation activity and nitric oxide (NO) production was examined. RM were induced in three week old specific pathogen free turkeys with Sephadex G-50, Thioglycollate broth, and an emulsion of incomplete Freund's adjuvant (IFA), supplemented either with Mycoplasma hyorhinis grown in Modified Channock broth (IFA-M. hyorhinis) or with Modified Channock broth (IFA-Broth). The RM were recovered by lavage of the lungs and air sacs and were purified by centrifugation through a Percoll suspension. Their oxygenation activity was evaluated in luminol-enhanced chemiluminescence assays, following stimulation with Zymosan A. The NO production was evaluated by incubating the RM with lipopolysaccharide (LPS) from Salmonella enteritidis for 24 or 48 hours. The number of recovered RM was slightly, but not significantly lower for Sephadex G-50 and IFA-Broth than for Thioglycollate broth and IFA-M. hyorhinis. RM elicited with Sephadex G-50 and IFA-Broth showed a significantly higher oxidative burst response to Zymosan A, compared to the Thioglycollate and IFA-M. hyorhinis elicited RM. Although all elicited RM showed a high NO production upon stimulation with LPS, no significant differences were seen in the NO production of the RM obtained following the different induction treatments. Our results point out that care should be taken when applying elicited RM for in vitro assays, as distinct levels of oxygenation activity were obtained using different induction protocols.

Animals↗
PubMed2002

Comparison of two methods for serotyping Ureaplasma urealyticum clinical isolates.

A newly developed enzyme linked immunosorbent assay (ELISA) method using monoclonal antibodies (MAbs) to the 14 serotypes of Ureaplasma urealyticum was compared to immunofluorescence assay (IFA) for serotyping U. urealyticum clinical isolates. Of the 102 vaginal isolates of U. urealyticum, five strains were lost and were excluded from analysis. Of the 97 strains analysed, a total of 86 (89%) strains were typeable by ELISA and a total of 89 (92%) strains were typeable by IFA. Eighty-six strains were typeable by both methods, three by IFA only and eight strains were not typeable neither by ELISA nor by IFA. Of the 86 strains typeable by both methods, complete concordance in serotyping results was found. The three strains not typeable by ELISA were typeable as serotype 4 by IFA. These three strains were reanalysed by ELISA after major modifications of the antigen preparation and were typeable as serotype 4. In conclusion, the ELISA was found suitable for serotyping clinical isolates. However, since the ELISA had a somewhat lower performance than IFA, strains not typeable by ELISA, should be retested by another technique such as IFA.

Antibodies, Bacterial↗
PubMed2002

Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA.

Flavivirus infections are a significant public health problem, since several members of the Flaviviridae family are highly pathogenic to humans. Accurate diagnosis and differentiation of the infecting virus is important, especially in areas where many flaviviruses are circulating. In this study we evaluated a newly developed commercially available immunofluorescence assay (IFA) (INDX, Baltimore, MD, USA) for the detection of IgM and IgG antibodies against dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. IFA was compared with standard diagnostic enzyme immunoassays (EIAs) specific for the detection of IgM and IgG antibodies against these viruses. Forty-seven serum samples from patients with a defined flavivirus infection were tested. As controls, serum samples from individuals with antibodies against tick-borne encephalitis virus and hepatitis C virus as well as healthy individuals were included. The results obtained from this study indicate that IFA showed a significantly better discrimination for flavivirus specific IgM antibodies than did the standard IgM specific EIAs (the overall cross-reactivity varied between 4 and 10% by IFA and 30-44% by EIA for the respective viruses). In contrast, the detection of flavivirus specific IgG antibodies showed high cross-reactions in both IFA and EIAs (overall cross-reactivity 16-71 and 62-84%, respectively). This study clearly stated the complexity of flavivirus diagnosis, showing that one cannot rely on one assay or search for one virus only. The flavivirus IFA is a useful tool for the identification of flavivirus infections during the acute stage of disease. In particular, IFA can be an important diagnostic tool for testing samples from travellers who have been accidentally exposed to these viruses.

Antibodies, Viral↗
PubMed2002

Induction of skewed Th1/Th2 T-cell differentiation via subcutaneous immunization with Freund's adjuvant.

CD4+ T cells differentiate into at least two distinct subsets, Th1 and Th2, that are characterized by their cytokine-producing profiles. In this study, we attempted to delineate whether and how CD4+ T-cell responses could be skewed in one direction or another. BALB/c mice were immunized with chicken ovalbumin (OVA) emulsified with either incomplete or complete Freund's adjuvant (IFA or CFA). When lymph node cells were assessed on day 7, antigen specific proliferation was similarly observed both in the mice immunized with IFA and CFA. In contrast, on day 28 there was a less significant response in the mice primed with IFA than in those primed with CFA. ELISA analyses revealed more Th1 predominant cytokine production by T cells immunized with OVA+CFA rather than in IFA, which resulted in balanced IFN-gamma and IL-4 production. Flow cytometric analyses of intracellular cytokines confirmed that T cells from mice primed with CFA produced Th1 cytokines more predominantly. When lymph node dendritic cells (DC) were compared for their co-stimulatory molecule expression, priming with CFA and IFA similarly upregulated CD80 and CD86 expression by lymph node DC, and no significant differences were observed in CD40, 54, 80 and 86 expression between the DC harvested from IFA and CFA immunized mice. In addition, both priming with IFA and CFA similarly induced IL-12 production by DC. Thus, although the reason(s) for the preferential induction of a Th1/Th2 response remains unknown, these results indicate that a relatively Th1/Th2 skewed response is differentially induced by different types of adjuvants, and induction of a Th1 skewed response may be responsible for long lasting cellular immunity.

Animals↗
PubMed1995

Inhibitory effect of ferulic acid and isoferulic acid on murine interleukin-8 production in response to influenza virus infections in vitro and in vivo.

We investigated the effect of ferulic acid (FA) and isoferulic acid (IFA), which are active components of the rhizoma of Cimicifuga species used frequently as anti-inflammatory drugs in Japanese Oriental medicines, on murine interleukin-8 (IL-8) production in response to influenza virus infections in vitro and in vivo by antibody-sandwich enzyme-linked immunosorbent assay. In the in vitro study, the murine macrophage cell line RAW 264.7 was infected with influenza virus at a dose of 10 plaque forming units (PFU)/cell and cultured in the presence or absence of drugs. Both FA and IFA reduced the IL-8 levels in the 20-h conditioned medium in comparison with control in a dose-dependent manner. The effect of IFA was greater than that of FA: IL-8 levels were reduced to 43% and 56% of the control in the presence of 100 micrograms/ml of IFA and FA, respectively. In the in vivo study, mice were infected with 1,000 PFU of virus and received daily oral administrations of Cimicifuga heracleifolia extract (5 mg/mouse/day), FA (0.5 mg/mouse/day), IFA (0.125 mg/mouse/day), or phosphate buffered saline. The three drugs showed a tendency to reduce IL-8 levels in bronchoalveolar lavage (BAL) obtained 2 days after infection. Moreover, both FA and IFA also significantly reduced the number of exuded neutrophils into BAL. However, the drug administrations did not affect the virus yields in BAL. These data suggest that FA and IFA are novel and potent inhibitors of murine IL-8 production and might act as one of the main components of anti-inflammatory rhizoma of Cimicifuga species.

Animals↗
PubMed1990

Epidemiologic implications of limited-duration seropositivity after toxoplasma infection.

In the Netherlands, the prevalence of immunoglobulin G antibodies against Toxoplasma shows a plateau at older ages. The titer distributions in the indirect fluorescent antibody (IFA) test do not change after the age of 40 years. In recent serologic studies, it has been observed that enzyme-linked immunosorbent assay of toxoplasma antibodies yields much higher seroprevalence values at older ages. These findings indicate that IFA seropositivity is not lifelong. It has been suggested in the literature that the IFA data in the older age groups reflect a dynamic balance between reversions to seronegativity and conversions to seropositivity. In this paper, the epidemiologic implications are examined by means of a mathematical model. The seroprevalence data linked to the model indicate that the mean duration of IFA seropositivity after infection is 40 years and that the annual infection risk in the reproductive age group (15-40 years) is 4-6%. A further implication of a limited duration of IFA seropositivity is that a certain fraction of IFA-seronegative persons have experienced an infection in the past. The model-based analysis indicates that the predictive value of a negative IFA test is very low at the older ages. The model is consistent with the available data, and it provides a framework for further theoretical and data-related research.

Adolescent↗
PubMed2004

Distinctive patterns of autoimmune response induced by different types of mineral oil.

Although mineral oils are generally considered nontoxic and have a long history of use in humans, the mineral oil Bayol F (incomplete Freund's adjuvant, IFA) and certain mineral oil components (squalene and n-hexadecane) induce lupus-related anti-nRNP/Sm or -Su autoantibodies in nonautoimmune mice. In the present study, we investigated whether medicinal mineral oils can induce other types of autoantibodies and whether structural features of hydrocarbons influence autoantibody specificity. Female 3-month-old BALB/c (16-45/group) mice each received an i.p. injection of pristane (C19), squalene (C30), IFA, three medicinal mineral oils (MO-F, MO-HT, MO-S), or PBS. Sera were tested for autoantibodies and immunoglobulin levels. Hydrocarbons were analyzed by gas chromatography/mass spectrometry. IFA contained mainly C15-C25 hydrocarbons, whereas MO-HT and MO-S contained C20-C40, and MO-F contained C15-C40. Pristane and n-hexadecane were found in IFA (0.17% and 0.10% w/v, respectively) and MOs (0.0026-0.027%). At 3 months, pristane and IFA induced mainly IgG2a, squalene IgG1, and MOs IgG3 and IgM in sera. Anti-cytoplasmic antibodies were common in mice treated with MO-F, as well as those treated with pristane, squalene, and IFA. Anti-ssDNA and -chromatin antibodies were higher in MO-F and MO-S than in untreated/PBS, squalene-, or IFA-treated mice, suggesting that there is variability in the induction of anti-nRNP/Sm versus -chromatin/DNA antibodies. The preferential induction of anti-chromatin/ssDNA antibodies without anti-nRNP/Sm/Su by MO-S and MO-F is consistent with the idea that different types of autoantibodies are regulated differently. Induction of autoantibodies by mineral oils considered nontoxic also may have pathogenetic implications in human autoimmune diseases.

Alkanes↗
PubMed2001

Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi.

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals.

Antibody Specificity↗
PubMed2006

Evaluation of Vircell enzyme-linked immunosorbent assay and indirect immunofluorescence assay for detection of antibodies against Legionella pneumophila.

We evaluated the abilities of the Vircell immunoglobulin G (IgG) and IgM indirect immunofluorescence assay (IFA) for Legionella pneumophila serogroup 1, the IgM and IgG enzyme-linked immunosorbent assay (ELISA) for Legionella pneumophila serogroup 1, and the IgM-plus-IgG ELISA for Legionella pneumophila serogroups 1 to 6 to diagnose Legionnaires' disease (LD) in a well-described sample of patients with and without LD. Also, we determined the agreements, sensitivities, and specificities of the different Vircell assays in comparison to a validated ELISA (Serion classic ELISA). Clinical sensitivity and specificity were 74.6% and 96.6%, respectively, for the IgM IFA, 65.1% and 88.0% for the IgG IFA, 92.3% and 100% for the IgM ELISA, 43.3% and 96.6% for the IgG ELISA, and 90.8% and 100% for the IgM-plus-IgG ELISA. Compared to Serion classic ELISA, agreement, sensitivity, and specificity were 80.0%, 83.1%, and 78.4%, respectively, for the IgM IFA, 75.2%, 66.0%, and 79.5% for the IgG IFA, 89.5%, 82.0%, and 97.6% for the IgM ELISA, 81.9%, 88.9%, and 78.0% for the IgG ELISA, and 93.5%, 90.0%, and 96.6% for the IgM-plus-IgG ELISA. The value of a positive diagnostic result obtained by the Vircell IgM IFA, the Vircell IgG IFA, and the Vircell IgG ELISA might not be acceptable for a diagnostic assay. Both the high specificities and sensitivities of the Vircell IgM ELISA and the IgM-plus-IgG ELISA and the high correlation with the Serion classic ELISA indicate that they are useful in the diagnosis of LD.

Adolescent↗
PubMed1981

Detection of rubella antibodies by hemagglutination inhibition, indirect fluorescent-antibody test, and enzyme-linked immunosorbent assay.

Using hemagglutination inhibition (HAI) as a reference method, 292 (40 nonimmune, 252 immune) human serum samples were tested by indirect fluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) for immune status and quantitation of rubella antibodies. The overall agreement with HAI for immune status was 99.7% (291/292) with IFA and 98.6% (288/292) with ELISA. Two specimens (0.7%, 2/292), negative by HAI, were equivocal by ELISA. Initially a 6.5% (19/292) overall disagreement was obtained for immune status evaluation between HAI and IFA, which was reduced to 0.3% (1/292) upon repeat testing. All of these samples were near the immune/nonimmune cutoff point (95 samples), reflecting an initial disagreement of 20% (19/95) in this category (HAI titers less than 1:20). Likewise, an initial overall disagreement of 4.5% (13/292) was obtained between HAI and ELISA which was reduced to 0.7% (2/292) upon repeated testing. Eleven of the 13 samples were near the immune/nonimmune cutoff point, reflecting an initial disagreement of 11.6% (11/95) with sera having an HAI antibody titer of less than 1:20. Quantitation of rubella antibodies by IFA showed an overall correlation with HAI of 86.6% within less than twofold titer and 99.3% within less than fourfold titers. In testing the ability of ELISA to quantitate antibody, a correlation coefficient (r) of 0.996 was obtained by plotting the measured average optical density (405 nm) of ELISA against the corresponding log of HAI titer. Both IFA and ELISA showed good correlation with HAI for immune status evaluation and for quantitation of rubella antibodies. Technically the HAI was the most cumbersome to perform, whereas IFA was the least technically demanding. Originally, 308 samples were tested; 16 samples (5.2%) could not be evaluated by IFA because of a high level of nonspecific fluorescence. The strict requirement of controlling the temperature range (23 to 24 degrees C) during substrate hydrolysis proved to be a problem with the ELISA test in our laboratory.

Antibodies, Viral↗
PubMed1988

Detection of respiratory syncytial virus in clinical specimens by viral culture, direct and indirect immunofluorescence, and enzyme immunoassay.

We evaluated prospectively the detection of respiratory syncytial virus (RSV) by culture and by direct antigen detection using an indirect immunofluorescence assay (IFA), a direct monoclonal immunofluorescence assay (DFA), and a monoclonal enzyme immunoassay (EIA). Of 221 specimens, 95 (43%) were culture positive for RSV, 4 (1.8%) contained more than one virus, and 17 (7.6%) contained a virus other than RSV. Overall, HEp-2 and Flow 6000 cells grew significantly more RSV isolates (82 and 72%, respectively) than A549 cells, which grew only 29% of the isolates. The mean time for RSV detection with HEp-2 cells was 2.9 days. This was significantly less than the mean time for RSV detection with either Flow 6000 cells (6.1 days) or A549 cells (6.4 days). Of 221 specimens, 129 were tested simultaneously by culture, IFA, and DFA. Of these 129 specimens, 62 (48%) were positive by culture, 69 (53%) were positive by IFA, and 70 (54%) were positive by DFA. For 92 specimens screened simultaneously by culture, IFA, and EIA, positive results were obtained for 33 (36%) of the specimens by both culture and IFA and for 29 (32%) of the specimens by EIA. Of 126 culture-negative specimens, 21 (17%) were positive for RSV when determined by IFA. Conversely, 14 (15%) of 95 RSV culture-positive specimens were negative by IFA, whereas DFA missed 19% of the culture-positive specimens. Compared with culture, the Kallestad EIA kit had a sensitivity and specificity of 73 and 92% respectively, but missed 9 (27%) of 33 culture-positive specimens. These data demonstrate that isolation by culture continues to be important for viral diagnosis of REV infections and that for valid comparative studies between viral isolations and rapid detection methods, both sensitive host cells and appropriate test conditions are required.

Cell Line↗
PubMed1989

Immunofluorescence assay for detection of antibodies to human immunodeficiency virus type 2.

A total of 215 serum samples were tested for antibodies against human immunodeficiency virus type 2 (HIV-2) with an immunofluorescence assay (IFA). Some samples originated from Denmark and some originated from Guinea-Bissau. The IFA results were compared with enzyme-linked immunosorbent assay (ELISA) and Western (immuno-) blot (WB) results. Twenty-nine serum samples were found to be true positive for HIV-2 antibodies as judged from WB and radioimmunoprecipitation results; all of these were also found to be positive in the HIV-2 IFA. Of 80 serum samples originating from HIV-1-infected persons, 60% showed reactivity in the HIV-2 ELISA, and 51% cross-reacted with at least one band in the HIV-2 WB. None of the sera cross-reacted in the HIV-2 IFA. A total of five serum samples (three African and two Danish) gave unspecific results in the HIV-2 IFA. It is concluded that the HIV-2 IFA is more specific and at least as sensitive as a first-generation ELISA and that IFA is superior to WB in discriminating between HIV-1 and HIV-2 infections.

Blotting, Western↗
PubMed1989

Detection of Cryptosporidium oocysts in human fecal specimens by an indirect immunofluorescence assay with monoclonal antibodies.

An indirect fluorescence assay (IFA) with monoclonal antibodies developed for the detection of Cryptosporidium oocysts in fecal smears (Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared with the Zeihl-Neelsen-modified acid-fast stain (MAFS) in 119 human fecal specimens collected between 1984 and 1987. The sensitivity of the IFA was 100%; all 56 specimens positive by MAFS exhibited fluorescence. There were 63 specimens negative for Cryptosporidium sp. by MAFS; of these, 61 were negative by IFA (97% specificity). This discrepancy may reflect an increased sensitivity of the IFA to detect oocysts that were not visualized by MAFS because of faint staining or a paucity of organisms. On average, the IFA required less time than the MAFS (1 versus 5 min, respectively) when only rare or few oocysts were present. Cost comparison of reagents showed the IFA to be three times more expensive. The IFA offers a reasonable alternative to the MAFS because of its high sensitivity and specificity, the simplicity of performing it and interpreting results, and its capability of providing a definitive diagnosis of Cryptosporidium oocysts.

Animals↗
PubMed1976

Diagnosis of herpes simplex virus infection by immunofluorescence.

The utility of the indirect immunofluorescent antibody (IFA) technique for diagnosis of herpes simplex virus (HSV) infection was examined by testing specimens for this agent from 31 patients with encephalitis or meningitis, 17 with conjunctivitis, 19 with genital disease, and 1 with genital disease and meningitis. Brain biopsy tissue from four patients with encephalitis was positive by IFA and virus culture for HSV. Leukocytes in cerebrospinal fluid from these four patients and one with HSV meningitis were also positive by IFA, but virus isolation attempts on the fluid were all negative. Conjunctival scrapings from two patients with conjunctivitis were positive for HSV by both IFA and virus culture. Eleven of 12 culture-positive lesions of herpes progenitalis were positive by IFA, and 1 dark field-positive syphilitic chancre was also positive for HSV by both IFA and culture. Evidence for specificity of the results was provided by internal controls in each test and negative results from patients with other diagnoses. Thus, the IFA technique constituted a rapid, sensitive, and specific diagnostic method for the diagnosis of HSV infections.

Brain↗
PubMed1993

Application of indirect immunofluorescence to detection of Dientamoeba fragilis trophozoites in fecal specimens.

An indirect fluorescent-antibody (IFA) assay was carried out to examine for the presence of Dientamoeba fragilis trophozoites in preserved fecal specimens. Antiserum to D. fragilis trophozoites was raised in a rabbit with a dixenic culture of D. fragilis (ATCC 30948) from the American Type Culture Collection. After absorption with Klebsiella pneumoniae and Bacteroides vulgatus, the immune rabbit serum was used for examination by the IFA assay. A total of 155 clinical samples were tested; 42 with no parasites, 9 with D. fragilis, and 104 with other parasites. The IFA assay identified seven D. fragilis organisms. Two specimens with doubtful IFA assay readings showed very scanty amounts of D. fragilis trophozoites on stained smears. There were no false-positive IFA assay readings. The IFA assay appeared to be a promising method because of its speed in screening. The specificity of the IFA assay indicates that other diagnostic tests such as an enzyme-linked immunosorbent assay could be developed to identify D. fragilis antigens in fecal specimens.

Animals↗