Rapid immunofluorescence microscopy for diagnosis of melioidosis

An immunofluorescent (IF) method that detects Burkholderia pseudomallei in clinical specimens within 10 min was devised. The results of this rapid method and those of an existing IF method were prospectively compared with the culture results for 776 specimens from patients with suspected melioidosis. The sensitivities of both IF tests were 66%, and the specificities were 99.5 and 99.4%, respectively.     

Comparison of detection of antibody to the acquired immune deficiency syndrome virus by enzyme immunoassay, immunofluorescence, and Western blot methods.

There was 100% agreement between enzyme immunoassay (EIA) (Abbott Laboratories), Western blot, and indirect immunofluorescence (IF) when these three methods were used to measure antibody to the acquired immune deficiency syndrome (AIDS) virus in sera from 142 high-risk individuals, indicating that IF was a sensitive alternative method for detecting antibody to this agent. Thirty-two (64%) of 50 EIA-positive plasma specimens from a blood bank and 6 (21%) of 28 EIA-positive sera from alternative testing sites were negative by IF. In addition, two EIA-negative sera from the latter group were positive by IF. Western blotting agreed with IF on those 40 specimens which gave discrepant results by EIA and IF. The IF method was determined to be equal to Western blotting in sensitivity and specificity for detection of AIDS antibody, and it was found to be useful for confirming positive EIA results, especially in specimens from individuals in low-risk groups.     

Cytomegalovirus detection by biotinylated DNA probes

Clinical specimens from 317 patients suspected of cytomeglovirus infection were examined by immunofluorescence (IF) using monoclonal antibodies and by a biotinylated DNA probe kit after cell culture isolation. Of the 317 samples, 68 were positive by culture isolation. Of these 67 were IF positive when the cytopathic effect (CPE) was 1⁺ or less, whereas 56 gave positive results with DNA probes when the CPE was 2⁺. A further 83 specimens were examined directly by immunoperoxidase histopathology (IHP), IF and the DNA probe kit: 26 of these were positive by IHP examination, 25 by IF and only 6 by DNA probes. The sensitivity of the DNA probe kit was not satisfactory when the clinical tissue specimens were directly examined. However, the sensitivity improved considerably to 82% if the specimens were propagated first in cell culture. The IF method detected the virus before and after cell culture isolation equally well (96%–98.5%). Compared to the IF method, the DNA probe kit is costly and requires more labor and time.This paper was presented in part at the 88th annual meeting of the American Society for Microbiology, Miami Beach, FL, USA, 1988     

Comparison of directigen RSV with viral isolation and direct immunofluorescence for the identification of respiratory syncytial virus.

An enzyme immunoassay membrane test (Directigen RSV) for the detection of respiratory syncytial virus in clinical specimens was compared prospectively with isolation in cell culture and direct immunofluorescence (IF). A total of 315 nasopharyngeal wash specimens from pediatric patients were examined. Directigen RSV was 86.1% sensitive and 91.3% specific for specimens positive by isolation in cell culture and/or IF, with 88.6% agreement. The false-positive rate was 16%; 2 of 20 specimens giving false-positive reactions by Directigen RSV were true-positives by blocking assay. Twenty-seven specimens (8.5%) whose results were initially uninterpretable by Directigen RSV due to filtration difficulties were diluted and upon retesting produced acceptable results. Sixty-three viral isolates and/or IF identifications of virus antigens representing seven virus groups other than respiratory syncytial virus were also found; cross-reactions between Directigen RSV and other viruses were not observed. Directigen RSV will be useful as an immediate procedure and in facilities lacking a comprehensive virology laboratory.     

Direct immunofluorescence staining for detection of herpes simplex and varicella-zoster virus antigens in vesicular lesions and certain tissue specimens.

Direct immunofluorescence (IF) staining was compared with virus isolation for detection of herpes simplex viruses (HSV) and varicella-zoster virus (VZV) directly in clinical materials. These included 199 vesicular lesion specimens and 280 tissue specimens. Correspondence between IF and isolation results was 88% in testing for HSV in lesion specimens and 98% in testing for HSV in various tissue (mostly brain) specimens. Overall, IF was positive for 82% of the specimens in which HSV was demonstrated, and virus was isolated from 89% of the HSV-positive specimens. IF was markedly more sensitive than isolation for demonstrating VZV in lesion and tissue specimens, detecting all of the specimens positive for VZV, whereas isolation detected only 23%. IF detected VZV antigen in a number of lesion specimens taken late after onset, past the time when they would be expected to yield infectious virus. Specificity of positive IF reactions for HSV or VZV in the absence of virus isolation was supported by the facts that (i) staining was obtained with only a single, presumably homologous, immune conjugate, (ii) clinical symptoms were compatible with infection with the virus for which positive IF findings were obtained, and (iii) positive electron microscopy findings for herpesviruses or positive serological results for VZV were also obtained in some instances. Factors to be considered in achieving specificity of IF staining for these human herpesviruses are discussed.     

Comparison of indirect immunofluorescence and Western blot for detection of anti-human immunodeficiency virus antibodies.

There was 100% agreement between the results of indirect immunofluorescence (IF) and Western blot testing when these methods were used to detect antibodies to the human immunodeficiency virus in sera from 25 patients with acquired immune deficiency syndrome (AIDS), 20 patients with AIDS-related complex, 186 subjects at high risk for AIDS, and 40 healthy heterosexuals. However, there was only an 88.7% correlation between IF and Western blot results for 728 sera from blood and plasma donor centers that were selected on the basis of screening enzyme immunoassay reactivity. IF tests yielded nine false-negatives and were equivocal, yielding a nonspecific pattern of reactivity for both infected and uninfected cells for 73 of these specimens. The IF and Western blot methods were equal in performance for the detection of anti-human immunodeficiency virus antibodies in the high-risk and unselected low-risk groups, proving to be a practical approach for testing specimens from these subjects. However, the Western blot was the most acceptable method for the validation of specimens from groups at low risk for AIDS that were selected based on enzyme immunoassay reactivity.     

Comparison of Directigen FLU-A with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza A virus.

Directigen FLU-A, an enzyme immunoassay membrane test, was compared prospectively to isolation in cell culture and direct immunofluorescence (IF) for the detection of influenza A virus. One hundred ninety specimens were evaluated by Directigen FLU-A and cell culture; 184 of these specimens were also tested by direct IF. The sensitivity of Directigen FLU-A compared to isolation in cell culture and direct IF was 100%. The specificities of Directigen FLU-A compared to isolation and direct IF were identical, 91.6%. Fourteen specimens that were positive by Directigen FLU-A did not yield virus in culture; two of the specimens, however, were positive by direct IF, and four other specimens were not specimens of choice for the test. A positive Directigen result had positive predictive values of 62.6 and 75.0% compared to isolation and direct IF, respectively; a positive Directigen result with an intensity reading of 2+ or greater, however, had positive predictive values of 85 and 100% compared to isolation and direct IF, respectively. In all comparisons, the negative predictive value was 100%. There was no evidence that cross-reactivity occurred with non-influenza A antigens. Directigen FLU-A should serve as a convenient screening test for influenza A and as a rapid test supported by isolation in cell culture during an influenza outbreak.     

Comparison of immunofluorescence, enzyme immunoassay, and Western blot (immunoblot) methods for detection of antibody to human T-cell leukemia virus type I.

A total of 3,349 serum samples were screened by the immunofluorescence (IF) method for antibody to human T-cell leukemia virus type I (HTLV-I). Only 9 of 2,409 specimens from selected individuals, blood bank donors, patients with encephalitis-meningitis, and human immunodeficiency virus antibody-positive homosexual or bisexual men were reactive by IF. In addition, 940 serum samples from intravenous drug abusers were tested by IF and also by an HTLV-I enzyme immunoassay (EIA) method. Of these, 222 (24%) were positive for both HTLV-I and HTLV-II antigens by IF, and 191 of these 222 were also reactive in the HTLV-I EIA. Of the 31 IF-positive, EIA-negative serum samples, 20 exhibited optical density readings greater than or equal to 70% of the positive cutoff in the EIA, and 29 samples reacted with 1 or more bands in the Western blot (immunoblot) test. An additional 10 specimens that were EIA negative reacted only with HTLV-I by IF. Differences in staining morphology and in reactions on HTLV-I and HTLV-II antigens before and after absorption of the serum specimens with HTLV-I and HTLV-II-infected cell pellets revealed six distinct serological patterns by IF. These results indicate that infections by HTLV-I or by another closely related retrovirus(es) occur in California. Further studies utilizing statistically valid sampling methods are needed to estimate true prevalence rates among various groups. IF and Western blot tests should supplement the EIA method to maximize sensitivity and specificity of test procedures.     

Comparison of immunofluorescence and culture for the detection of Actinomyces israelii in wearers of intra-uterine contraceptive devices

A direct immunofluorescence (IF) method was compared with traditional culture methods for the detection of Actinomyces israelii in endocervical and intra-uterine-device (IUD) smears from 124 IUD wearers. Of 11 specimens that gave positive results by IF, only one was positive by culture. Of the 10 patients with positive IF specimens, three (30%) had signs and symptoms suggestive of pelvic infection and no other pathogen was detected. Direct IF of cervical smears offers a simple, relatively cheap method to screen IUD wearers for A. israelii. Clinical management of such cases is discussed.     

Evaluation of the ESPLINE® Influenza A &; B-N assay for the detection of influenza A and B in nasopharyngeal aspirates

Several direct antigen tests for the detection of influenza often lack sensitivity compared to immunofluorescence (IF) on the specimens and viral culture (VC). We evaluated the performance of a rapid test, the ESPLINE® Influenza A &; B-N assay. A total of 302 respiratory specimens were collected at the University Hospital of Antwerp. A first group of 60 samples taken during the H1N1 outbreak (2009–2010) and a second group of 242 samples stored during the seasonal influenza epidemics (2000–2009) were analyzed with the ESPLINE® test. A subset of samples were also evaluated with the BinaxNOW Influenza and the Clearview Exact Influenza. The results were compared to IF on the specimens, VC with IF, and the combination of both, which was considered as the gold standard. The ESPLINE® test’s overall sensitivity and specificity were 91% and 97%, during the H1N1 season 80% and 93%, and for the detection of seasonal influenza 93% and 97%, respectively. In comparison to the BinaxNOW Influenza and the Clearview Exact Influenza, all tests demonstrated a similar specificity of 92.0–100% but a significantly different sensitivity of 44.4–86.0%, with the ESPLINE® test being significantly more sensitive. Due to its very good performance and simplicity, the ESPLINE® test facilitates urgent testing. The test seems less sensitive to detect H1N1 compared to seasonal influenza, although the difference is borderline not significant (p?=?0.067).     

A MOLECULAR METHOD FOR TYPING HERPES SIMPLEX VIRUS ISOLATES AS AN ALTERNATIVE TO IMMUNOFLUORESCENCE METHODS

Background: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. Study design: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase (pol) gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.     

Detection of viral and chlamydial antigens in open-lung biopsy specimens

The recovery of viruses and Chlamydia trachomatis from cell cultures and the detection of their antigens in impression smears prepared from open-lung biopsy (OLB) specimens from immunocompromised adults were compared. Touch impression smears were prepared on three slides, each containing eight wells. OLB tissue was homogenized (Stomacher) and inoculated into MRC-5, primary monkey kidney, and McCoy cell cultures. The direct and indirect immunofluorescence (IF) tests were used to detect antigens to the following organisms: herpes simplex virus, adenovirus, parainfluenza types 1 and 3, respiratory syncytial virus, cytomegalovirus (CMV), Chlamydia trachomatis, influenza types A and B, and varicella-zoster virus. Of 105 OLB specimens, 21 viral isolates (20%) were recovered in cell culture; 20 were CMV and one was an influenza virus type A (H3N2). Both culture and IF results were positive with 12 specimens, but in nine instances a virus was isolated and IF was negative or eight times culture results did not yield the organism but IF test results were positive. Chlamydia trachomatis was never isolated or detected by IF. The authors recommend that for optimal detection of CMV from OLB specimens a new rapid centrifugation-enhanced cell culture system be used in preference, or in conjunction with a preliminary IF screen of impression smears for CMV detection.     

Immunofluorescence staining for detection of variola virus.

In September 1975 Bangladesh was the only country in the world with endemic variola major, and the eradication of the disease was imminent. A rapid and accurate laboratory diagnostic method was required to supplement immunodiffusion in agar gel and culture on chorioallantoic membrane of embryonated egg available at the Institute of Public Health in Dacca, Bangladesh. To determine its effectiveness, a new, improved immunofluorescence (IF) staining technique was introduced. Laboratory specimens (scabs or vesicular or pustular impressions) were collected from patients who had, or were suspected of having, smallpox. Seventy-eight of 144 specimens collected were found to be IF positive for smallpox. As the number of laboratory-positive cases far exceeded the number of clinically diagnosed smallpox cases, IF-positive cases were reinvestigated and subsamples of the IF-positive specimens were tested at a World Health Organization poxvirus reference laboratory at the Centers for Disease Control in Atlanta, Ga. The results indicated 100% sensitivity for the IF technique (no false-negative results) in diagnosing variola major but also showed a high rate of false-positive results. Consequently, IF could not be recommended as a routine screening test for smallpox.     

Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods.

To determine the minimum number of Cryptosporidium oocysts that can be detected in stool specimens by diagnostic procedures, stool samples seeded with known numbers of Cryptosporidium parvum oocysts were processed by the modified Formalin-ethyl acetate (FEA) stool concentration method. FEA concentrates were subsequently examined by both the modified cold Kinyoun acid-fast (AF) staining and fluorescein-tagged monoclonal antibody (immunofluorescence [IF]) techniques. Oocysts were more easily detected in watery diarrheal stool specimens than they were in formed stool specimens. For watery stool specimens, a 100% detection rate was accomplished at a concentration of 10,000 oocysts per g of stool by both the AF staining and IF techniques. In formed stool specimens, 100% of specimens seeded with 50,000 oocysts per gram of stool were detected by the IF technique, whereas 500,000 oocysts per g of stool were needed for a 100% detection rate by AF staining. Counting of all oocysts on IF slides indicated a mean oocyst loss ranging from 51.2 to 99.6%, depending on the stool consistency as determined by the FEA concentration procedure. Our findings suggest that the most commonly used coprodiagnostic techniques may fail to detect cryptosporidiosis in many immunocompromised and immunocompetent individuals.     

Rapid viral diagnosis of acute respiratory infections: comparison of enzyme-linked immunosorbent assay and the immunofluorescence technique for detection of viral antigens in nasopharyngeal secretions.

Nasopharyngeal secretions from adults and children were obtained in Stockholm, Sweden, for routine diagnosis of influenza A virus, influenza B virus, respiratory syncytial (RS) virus, parainfluenza type 3 virus, and adenovirus infections by demonstration of viral antigens directly in the specimens. The cells in nasopharyngeal secretions were pelleted by centrifugation for preparation of cell deposits for diagnosis by the immunofluorescence technique (IF) in London, England, and in Stockholm, whereas the supernatants were used to diagnose infection by the enzyme-linked immunosorbent assay (ELISA) in Stockholm. Titrations of the various purified viruses showed that ELISA could detect viral antigens in amounts corresponding to 1 to 10 ng of virus protein per test well. In a series of 73 specimens tested for influenza A, RS, and parainfluenza type 3 viruses by IF in London and by ELISA in Stockholm, 15 of 18 RS, 14 of 15 influenza A, and 2 of 2 parainfluenza type 3 viral infections were diagnosed by ELISA as compared with IF, giving sensitivities for RS and influenza A viral diagnosis of 83 and 93%, respectively, and a specificity of 100%. In another series of specimens from 35 patients tested for influenza B virus and adenovirus, five influenza B virus and four adenovirus infections were diagnosed by both methods; one additional influenza B infection was detected only by IF and another only by ELISA. Comparisons of diagnostic results between the two methods performed in Stockholm gave nonagreement of results for 37 of 1,593 tests (2.5%) for the five viruses. The conclusion reached was that the described ELISA, although a satisfactory test, had somewhat less sensitivity than did IF for the detection of respiratory viral infections. This could possibly be explained by unnecessary dilutions of specimens at the time of collection; transportation, processing, and storage of specimens were less complicated than for IF.     

Rapid detection of varicella-zoster virus in clinical specimens using monoclonal antibodies on shell vials and smears

Monoclonal antibodies were used for detecting varicella-zoster virus (VZV) by immunofluorescence (IF) in clinical specimens inoculated on shell vial cultures. Vesicles of 74 patients with varicella or herpes zoster-like eruptions were tested by this method and by conventional cell culture. Further diagnostic tests were direct IF on smears (42 patients), the cytological Tzanck test (28 patients), and serology (30 patients). Both IF assays were performed with the commercial VZV-specific monoclonal antibody 3B3 (Ortho). Using the shell vial technique, we found VZV in 37 (50%) of the patients, on average after 3 days. The conventional culture method yielded 26 positives (35%), on average after 7.5 days. Twenty-nine of the shell vial IF-positive patients were also positive by direct IF on smears (30 tested), while 15 gave positive Tzanck smears (18 tested). The sensitivities of the shell vial IF test, the direct IF test, the conventional culture method, and the Tzanck test were about 95%, 97%, 70%, and 79%, respectively. For the specificities, we found at least 97% for the shell vial IF test, at least 91% for the direct IF test, and about 78% for the Tzanck test. We conclude that both VZV IF tests are much more reliable than the conventional cell culture method and the Tzanck test for the laboratory diagnosis of VZV infections.     

Early detection of cytomegalovirus in cell culture by a monoclonal antibody

A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytopathogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70 degrees C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods.     

The detection of respiratory syncytial virus in nasopharyngeal aspirates: assessment, formulation, and evaluation of monoclonal antibodies as a diagnostic reagent

Comparisons were made between standard methods of cell culture, indirect immunofluorescence (IF) using hyperimmune respiratory syncytial virus (RSV) antiserum, and indirect IF using mouse monoclonal antibodies directed against various epitopes of RSV for the detection of RSV in nasopharyngeal aspirates. The monoclonal antibodies were used singly and in pools of different specificities which in turn were tested in both direct and indirect IF. In a preliminary study, aspirates from 227 infants were examined for RSV by standard methods. The results were compared with the detection of RSV in these aspirates using nine separate monoclonal antibodies and a pool consisting of five monoclonal antibodies. Respiratory syncytial virus was detected in 64 (28%) by cell culture, in 68 (30%) by indirect IF using bovine polyclonal antibody (BPA), and in 75 (33%) by indirect IF using the monoclonal antibody pool. The nine individual monoclonal antibodies when tested separately were less sensitive, detecting between 8 and 77% of all aspirates found to be positive by culture. After statistical analysis of the results obtained in the preliminary study, a refined monoclonal antibody pool was prepared and in a further study was tested by both direct and indirect IF in parallel with our two standard methods. Slides prepared from 303 nasopharyngeal aspirates collected between 1981 and 1984 and either tested the same day or stored at -20 degrees C were used to evaluate these reagents. Overall agreement between the four tests was found in 274 (90%) specimens. Cell culture detected RSV in 68 (22%) specimens, indirect IF with BPA in 67 (22%), indirect IF with monoclonal antibody in 72 (24%), and direct IF with monoclonal antibody in 79 (26%). The pool of monoclonal antibodies used in direct or indirect IF was thus more sensitive than our standard methods for the detection of RSV in nasopharyngeal aspirates, and direct IF tests could be completed in 40 minutes.