Immunofluorescence for routine diagnosis of respiratory syncytial virus infection

A comparison of immunofluorescent tests for the diagnosis of respiratory syncytial (RS) virus infections was carried out on 42 hospitalized cases of respiratory infection in childhood. Respiratory syncytial virus was detected in 22 (52%) cases, the most sensitive method of detection being by indirect immunofluorescence of Bristol HeLa tissue cultures inoculated with nasopharyngeal aspirates. The highest detection rate was in bronchiolitis cases (92%). Detection of antibody rises in paired sera, eight days apart, confirmed RS virus infection in 13 of 16 cases, the most sensitive test being detection of a specific rise in IgG antibody by indirect immunofluorescence. A serodiagnosis was made in all 10 non-bronchiolitis cases. Recommendations are made for the application ofimmunofluorescence to routine diagnosis of RS virus infection.     

Comparison of two new tests for rapid diagnosis of respiratory syncytial virus infections by enzyme-linked immunosorbent assay and immunofluorescence techniques.

The sensitivity and the specificity of two new commercial reagent tests, an indirect fluorescent antibody test (FAT) with a mouse monoclonal antibody (MAb) against respiratory syncytial virus (RSV) and an enzyme-linked immunosorbent assay (ELISA) RSV antigen detection kit, were determined by a comparison of results from these tests with those of tissue culture isolation and an indirect FAT with bovine polyclonal antibody (BPA). Of 251 nasal aspirates from infants with suspected RSV infection, positive results were found for 99 (39%) by the FAT-MAb, 93 (37%) by the FAT-BPA, and 87 (35%) by the ELISA; 69 of 240 (29%) were positive by cultures. The FAT-MAb was a more sensitive technique than cultures, with 87% sensitivity for the FAT-MAb and 84% for the ELISA. It was also more sensitive than the FAT-BPA, with 97% sensitivity for the FAT-MAb and 85% for the ELISA. This could be caused only by the distinctive volume of suspended specimens used in these tests. Of 171 negative culture specimens, positive (but not false-positive) results were found for 18% by the FAT-MAb and for 12% by the ELISA. Inversely, 13% of 69 culture positive specimens were FAT-MAb negative and 16% were ELISA negative, emphasizing the importance of tissue cultures for the maximum recovery of RSV, as well as for detection of other respiratory viruses. The FAT-MAb and ELISA were easy to perform and interpret, thus facilitating wider use.     

A rapid test for detection of respiratory syncytial virus in nasopharyngeal secretion

A new rapid membrane enzyme immunoassay (MEIA; Directigen RSV) for detection of respiratory syncytial virus (RSV) was evaluated using samples of nasopharyngeal secretion from infants and children with acute respiratory disease. The MEIA was compared with an immunofluorescent antibody (IF) technique using a sensitive biotin-avidin (BA) EIA as reference. Of 242 samples tested, 108 were positive by the MEIA and 123 by the BA-EIA. Of 144 samples which were also tested by the IF technique, 57 were positive by the BA-EIA and 43 by the IF technique. These results give a sensitivity of 86 % and 72 % for the MEIA and IF technique respectively. Of 57 samples found to be positive by the BA-EIA, 41 were positive by the IF technique, but 48 were positive by the MEIA. The MEIA is thus more sensitive than the IF technique but less sensitive than the BA-EIA in detecting RSV in nasopharyngeal secretions.     

Diagnostic efficacy of two rapid tests for detection of respiratory syncytial virus antigen.

With the availability of ribavirin therapy for serious respiratory syncytial virus (RSV) infections, rapid diagnostic tests for the detection of RSV antigen are increasingly important. Efficacies of a commercially available enzyme immunoassay (EIA) (Abbott Laboratories, North Chicago, Ill.) and a fluorescent-antibody assay (FA) were evaluated in a study involving 135 specimens from children with respiratory symptoms. A nasal wash specimen was cultured immediately on RSV-sensitive A549 cells; the nasal wash was also used for EIA. FA was performed on a nasopharyngeal swab specimen with bovine anti-RSV and anti-bovine immunoglobulin G antisera (Burroughs Wellcome Co., Research Triangle Park, N.C.). A total of 39 specimens (28%) were tissue culture positive, including 35 EIA-positive and 37 FA-positive samples (sensitivities, 90 and 95%, respectively). All 96 tissue culture-negative specimens were EIA negative (specificity, 100%); 94 of these 96 specimens were FA negative (specificity, 98%). Positive and negative predictive values for the tests were as follows: 100 and 96% for EIA, respectively, and 95 and 98% for FA, respectively. Other viruses, including influenza A virus, adenovirus, enterovirus, and herpes simplex virus, were isolated in nine cases. One adenovirus-positive specimen had a false-positive RSV FA result; all nine specimens were RSV EIA negative. Both tests performed well in our study and provide cost-effective alternatives to tissue culture. The RSV EIA, in particular, uses standard serologic techniques and equipment and does not require expertise in virology. More widespread availability of rapid diagnostic tests for RSV will hopefully result in early and appropriate use of antiviral therapy in patients at risk for serious RSV infections.     

Comparison of an immunochromatography test with multiplex reverse transcription-PCR for rapid diagnosis of respiratory syncytial virus infections

A new commercial rapid 10-min one-step immunochromatography (IC) test, SAS RSV test, was compared to another IC test, Directigen EZ RSV, employing RT-PCR as the "gold standard" for detecting respiratory syncytial virus. Of 102 clinical samples, 79 were positive by RT-PCR, 66 (82.5%) were positive with the SAS RSV test, and 55 (69.6%) were positive with Directigen EZ RSV. The specificity of the new test was 91.3% (21 of 23), similar to that of Directigen EZ RSV (100% [23 of 23]). This test performs well enough to be used for patient care.     

Rapid diagnosis of respiratory syncytial virus infection by using Pernasal swabs.

The sensitivity and specificity of direct immunofluorescence microscopy performed on Pernasal swab specimens and compared with those of nasopharyngeal aspirates were 93 and 99%, respectively. Posterior nasopharyngeal swabs applied immediately to microscope slides allow a rapid and simple screening procedure for the diagnosis of acute respiratory syncytial virus infections.     

Allergy skin test responses during experimental infection with respiratory syncytial virus.

BACKGROUND: Allergy skin testing is one of the most frequently performed physician office procedures. Many factors can affect the results of those tests, including the well-defined suppressive effect of systemic antihistamines. False-positive allergen skin test results are known to occur; however, contributing factors are not well understood. OBJECTIVE: To determine whether a viral upper respiratory tract infection affects allergy skin test responsiveness. METHODS: We performed skin tests with histamine and a panel of geographically relevant inhalant allergens on 16 adults before and 3, 6, and 21 days after experimental exposure to respiratory syncytial virus (RSV), a virus that causes signs and symptoms of a cold. RESULTS: The RSV exposure, with and without documented infection, caused increased wheal and flare areas to histamine and allergen and de novo positive allergen test responses in individuals with no measurable responses at baseline. These were noted as late as 21 days after RSV exposure and may be consistent with mediation by up-regulated neurogenic inflammation during RSV infection. CONCLUSION: These results may have implications for explaining the cause of such well-known complications of RSV infection as otitis media, bronchiolitis, and asthmatic exacerbation.     

Problems of laboratory diagnosis of respiratory syncytial virus infection in childhood

Summary During December, 1962 to March, 1963 the Glasgow area experienced an epidemic of RS virus infection amongst young children. Throat swabs for virus isolation and two or more serial specimens of sera were obtained from each of 42 children (41 under 3 years of age) with acute respiratory infections.A higher proportion of infections were diagnosed serologically by CF than by virus isolation, a four-fold or greater rising titre being obtained in 47% of the cases. However, two factors were of importance in obtaining optimal results by CF — namely (a) collection of convalescent serum after the fourteenth day of illness, preferably between the fourth and sixth weeks, and (b) the use of a potent antigen. Some difficulties in the preparation of high-titre antigen were studied.Higher neutralizing antibody levels were obtained in some cases against the current epidemic strain than against the prototype strain; however, they were generally much lower than the corresponding CF titres.For virus isolation throat swabs were inoculated directly after collection into tissue culture tubes in the ward. One major disadvantage of this method was that cultures from 12 swabs had to be discarded because of fungal contamination. Of the remaining 29, RS virus was isolated from 28%.     

Evaluation of the QuickLab RSV test, a new rapid lateral-flow immunoassay for detection of respiratory syncytial virus antigen

Rapid respiratory syncytial virus (RSV) diagnosis is vital to the prevention of nosocomial RSV infections. We evaluated a new rapid lateral-flow RSV immunoassay, the QuickLab RSV test, that requires use of only one reagent. We compared QuickLab to the Directigen RSV (DIR) assay, which requires six reagents, and direct fluorescent antibody (DFA) testing. DFA results were considered the "gold standard." For 133 nasopharyngeal aspirates tested, DFA results were 77 (57.8%) positive, 47 (35.3%) negative, and 9 (6.8%) indeterminate. The sensitivities, specificities, positive predictive values, and negative predictive values of QuickLab and DIR tests were 93.3% (70 of 75) and 80.8% (59 of 73), 95.6% (43 of 45) and 100.0% (46 of 46), 97.2% (70 of 72) and 100.0% (59 of 59), and 89.6% (43 of 48) and 76.7% (46 of 60), respectively. QuickLab was significantly (P = 0.02) more sensitive than DIR; the difference in specificities was not significant. DFA was more sensitive than DIR (P < 0.001) but not more sensitive than QuickLab (P = 0.45). The results of DIR testing were initially uninterpretable and required retesting with 15% of the specimens compared to 3% of QL results (P < 0.001). We conclude that the QuickLab RSV test has sensitivity similar to that of the DFA assay and better than that of the DIR assay. QuickLab testing is also simpler to perform and interpret than both DFA and DIR testing.     

Comparison of a new commercial enzyme immunoassay for rapid detection of respiratory syncytial virus

Two rapid methods for detection of respiratory syncytial virus in respiratory specimens were compared: direct immunofluorescence assay (DFA) with monoclonal antibody and an enzyme immunoassay (EIA) (Test-Pack RSV). Ninety-five nasopharyngeal washings and aspirates from 51 children were examined; the patients were hospitalized during a winter outbreak of RSV infection in the first trimester of 1990. A total of 41.0 % and 56.8 % of these samples were positive by EIA and DFA respectively. Considering only the 51 specimens collected at the onset of illness, EIA detected 72.5 % positive samples and DFA detected 78.4 %. In comparison with DFA, EIA was 92.5 % sensitive and 100 % specific for the acute phase of illness. When all the samples were taken into account, specificity was maintained but sensitivity fell to 72.2 %. The results show that both methods are useful during the acute phase of the illness, when the viral load is important. However, later on in the course of the infection DFA appears to be more sensitive than EIA.     

New immunochromatographic rapid test for diagnosis of acute Puumala virus infection

A new immunochromatographic rapid test, POC PUUMALA (Erilab Ltd., Kuopio, Finland), for detection of acute-phase Puumala virus (PUUV) infection was developed based on a highly purified baculovirus-expressed PUUV nucleocapsid protein antigen and lateral immunodiffusion techniques. After addition of sample (5 microl of serum, plasma, or fingertip blood) and buffer, PUUV-specific immunoglobulin M (IgM) antibodies, if present, together with the gold-conjugated anti-human IgM, formed a specific colored line in 5 min. The sensitivity and specificity of the test were evaluated with 200 serum samples and 30 fingertip blood samples. The reference method for the serum samples was a micro-capture enzyme immunoassay (EIA) for IgM and an immunofluorescence assay (IFA) for IgG antibodies. The analytical sensitivity and specificity of the rapid test were 100 and 99%, respectively, for unfrozen serum samples (n = 103; 12 PUUV IgM-positive samples). When freeze-thawed serum samples were used, the sensitivity and specificity were each 97.1% (n = 70; 35 PUUV IgM-positive samples). The specificity of the test was 96.2% for 27 serum samples with nonspecific IgM antibodies or rheumatoid factor (RF). The fingertip blood samples (n = 30) were negative, but they gave clear positive results when spiked with IgM-positive sera (n = 20). The results were in good agreement with the standard diagnostic methods. The rapid performance, the lack of need for refined laboratory equipment, and the high specificity with fresh serum and fingertip blood samples indicate that the developed POC PUUMALA rapid test is a useful tool for fast diagnosis of acute PUUV infection.     

The immunobiology of respiratory syncytial virus infection

There is increasing evidence that young children with severe respiratory syncytial virus (RSV)-induced bronchiolitis are at high risk of developing allergy and asthma during their later life. The determinants for this association are not well understood. Current studies suggest that both genetic backgrounds and unique characteristics of the virus play critical roles in determining the type of immune responses to RSV infection, leading to altered regulation of airway tone associated with wheezing. In susceptible subjects, RSV may either enhance the Th2 immune response or decrease the Th1 immune response. This altered Th1/Th2 cytokine response associated with RSV infection is not commonly observed among other RNA viruses, suggesting that RSV may have unique characteristics. Multiple clinical studies support the link between severe RSV bronchiolitis and the subsequent development of allergy and asthma. This link will be further tested by the ongoing large studies on the effect of early RSV intervention on the development of allergy. The administration of palivizumab, an anti-RSV monoclonal antibody, seems to be helpful for RSV prevention and treatment at early stage. There are no effective RSV vaccines available, and this is, at least in part, because of the poorly understood immunology and pathogenesis of RSV disease. The use of experimental animal models has led to a better, but not sufficient, understanding of the immunologic basis of RSV-induced disease, particularly asthma. Further studies on the immunopathology of RSV infection with animal models, including the nonhuman primate models, may help develop effective RSV vaccines.     

Viremia in respiratory syncytial virus infection]

Viremia was demonstrated to occur in experimental respiratory syncytial (RS) virus infection in suckling cotton rats and in natural infection in children. RS virus was isolated from the whole blood of the animals in 3 out of 6 experiments at 2, 5, 6, 7 and 15 days after inoculation, the maximum infectious titer being more than 10(4) TCPD50/0.1 ml. RS virus was also isolated from the blood of 7 out of 15 examined children presenting the typical clinical picture of RS virus disease during the epidemic season of RS virus infection. In 6 patients RS virus was isolated from one blood specimen at 1, 6 and 7 days after the onset, in one patient from 3 blood specimens at 6, 9 and 22 days after the onset. The demonstrated long-term persistence of virus in the blood suggests the possibility of existence of chronic RS virus infection.     

Primary respiratory syncytial virus infection in mice

A mouse model of respiratory syncytial virus (RSV) infection is described. A high-titered, large-volume inoculum results in replication of RSV to a high titer in lungs of BALB/c mice. Mice older than 15 weeks of age are more susceptible to RSV infection. Titers up to 10(6.9) plaque-forming units (pfu)/gram lung can be attained in 32-week-old mice. Older mice experience a clinical illness manifested by ruffled fur, reduced activity, and weight loss. Lung histology of older mice infected with RSV shows bronchiolitis and increased number of lymphocytes and macrophages in alveolar spaces compared with that of mice less than 8 weeks old. This model will serve as the basis for investigating immunodeterminants of recovery and protection from RSV infection.     

Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay.

Virus infectivity and antibody neutralization titers for respiratory syncytial virus were determined in cell cultures in microtiter plates. After an appropriate incubation period, the cells were fixed, and an enzyme-linked immunosorbent assay was performed directly in the microtiter plates for detection of virus. Results could be read and recorded automatically, which is especially helpful when running large numbers of tests.     

Detection of an immunoglobulin M response in the elderly for early diagnosis of respiratory syncytial virus infection.

The indirect fluorescent-antibody technique was compared with indirect and mu-capture enzyme-linked immunosorbent assays for the detection of respiratory syncytial virus (RSV) immunoglobulin M (IgM) in the elderly. Sera from 47 patients (mean age, 70 years) with acute lower respiratory tract infections caused by RSV were investigated. Specific IgM was detected in 81% (38 of 47) of the patients. The fluorescent-antibody technique, which gave 70% positive results, proved to be the most sensitive of the three methods. An IgM response was seldom seen in sera from the elderly within the first week of disease, but was present in 85% of sera (33 of 39) collected between days 11 and 30 of disease. In some patients it persisted for more than 6 weeks. Detection of IgM was found to be a useful tool for the diagnosis of RSV infections in elderly patients.     

Comparison of rapid immunofluorescence procedure with TestPack RSV and Directigen FLU-A for diagnosis of respiratory syncytial virus and influenza A virus.

A rapid immunofluorescence format requiring 20 min for completion was as effective as conventional indirect and direct immunofluorescence procedures for detecting respiratory syncytial virus and influenza A virus antigens in clinical specimens. Rapid immunofluorescence was more sensitive than TestPack RSV and comparable to Directigen FLU-A immunosorbent assays, which require 20 min for completion.     

The pathogenesis of respiratory syncytial virus infection in infant ferrets.

The infant ferret is susceptible to respiratory syncytial virus infection in both the upper and lower respiratory tracts. In the nose, viral replication is restricted to the surface respiratory epithelium in the nasal passages and turbinates. In the lungs, viral replication is of a lower order of magnitude and is localized in the alveolar cells. The pattern of viral replication in nasal tissues is independent of the age of the animal at infection, whereas the pattern in lung tissues shows a striking age dependence, with viral replication progressively decreasing as a function of age. Thes age dependence appears to be due to an intrinsic age-related mechanism yet to be defined. We feel that the infant ferret is an acceptable model for the study of respiratory syncytial virus disease and that the study of age dependence observed in ferrets may allow elucidation of the mechanisms involved in the age dependence seen in humans.