Evaluation of the anticomplement immunofluorescence test for detection of antibody to varicella-zoster virus.

The anticomplement immunofluorescence (ACIF) test was compared with complement fixation and fluorescent antibody to membrane antigen procedures for the detection of antibody to varicella-zoster virus. All of 50 sera from pregnant women contained antibody measured by ACIF (titer, greater than or equal to 1:10); only 27 (54%) were positive by complement fixation (P less than 0.01). For 16 paired sera obtained before and after varicella-zoster virus infection and tested by ACIF and fluorescent antibody to membrane antigen, the result agreed in 27 determinations (sensitivity, 94%; specificity, 81%). Of 99 sera submitted for routine determinations of immune status to the virus, 89 showed comparable results for both tests (sensitivity, 92.5%; specificity, 88%). The ACIF test offers a specific and sensitive alternative to the fluorescent antibody to membrane antigen procedure for the detection of antibody to varicella-zoster virus. In addition, the ACIF test is rapid, easy to perform, and uses commercially available reagents.     

Rapid diagnosis of varicella-zoster virus infection with a monoclonal antibody based direct immunofluorescence technique

Diagnosis of varicella-zoster virus (VZV) infection in immunocompromised patients is difficult because of the frequent atypical appearance. Accurate and early diagnosis is important to allow rapid commencement of antiviral chemotherapy, with consequent improvement in antiviral efficacy. A monoclonal based direct immunofluorescence antibody technique (VZV IFA) was assessed in parallel with viral culture in 56 patients with suspected VZV infection. A subgroup of 17 patients from this group with classical dermatomal herpes zoster all had positive VZV IFA tests. Only 6 patients (35%) were positive on viral culture. None of the 15 patients with proven herpes simplex virus infection had a positive VZV IFA, nor did any patient with positive VZV viral culture have a negative VZV IFA. The VZV IFA test is a rapid and sensitive technique for detecting infection with VZV.     

Comparison of anticomplement immunofluorescence and fluorescent antibody-to-membrane antigen tests for determination of immunity status to varicella-zoster virus and for serodifferentiation of varicella-zoster and herpes simplex virus infections.

The anticomplement immunofluorescence (ACIF) test was compared with the fluorescent antibody-to-membrane antigen (FAMA) test for determining varicella-zoster virus antibody levels as a measure of varicella-zoster virus immunity status. The ACIF test was found to be comparable to the FAMA test in sensitivity and could be used for examining sera at low dilutions of 1:2 and 1:4. In addition, the ACIF method proved to be a more economical procedure in terms of antigen required and personnel time necessary to perform the test. Heterologous varicella-zoster virus antibody titer rises were demonstrated by the FAMA test with 10 serum pairs from patients with clinically diagnosed genital herpes simplex virus infection, indicating that the FAMA test is no more suitable than other serological methods for serodifferentiation of those herpes simplex virus and varicella-zoster virus infections in which antibody increases occur to both antigens.     

Comparison of two methods for detecting varicella-zoster virus antibody with varicella-zoster virus cell-mediated immunity.

We evaluated an enzyme-linked immunoassay (EIA; BioWhittaker) and a latex agglutination (LA; Becton Dickinson) for varicella-zoster virus (VZV) antibody determination, using cell-mediated immunity (CMI) as a "gold standard." VZV EIA had a sensitivity, specificity, positive predictive value, and negative predictive value of 87, 91, 87, and 91%, respectively, compared with CMI. Correlation was excellent except when the varicella index was 0.9 to 1.2. We defined sera with varicella indices of 0.9 to 1.2 as indeterminate. LA had a sensitivity, specificity, positive predictive value, and negative predictive value of 96, 91, 97, and 90%, respectively, compared with EIA. LA reactivity only at a 1:2 dilution did not correlate with CMI, but sera reactive at dilutions of > or = 1:8 indirectly did. We defined indeterminate sera as those reactive at 1:2 and nonreactive at 1:8. EIA and LA were equivalent for determining VZV immune status, and both methods required modified criteria of interpretation to increase their specificity.     

Neutralizing antibody responses to varicella-zoster virus.

Neutralization of varicella-zoster (V-Z) virus by human sera and immune rhesus monkey sera was enhanced by fresh guinea pig complement. There was no marked difference in the degree to which complement enhanced neutralization by sera from current V-Z virus infections and sera from long-past varicella infections. Immunoglobulin G neutralizing antibody in sera from varicella cases was enhanced by complement to a slightly higher degree than was immunoglobulin M (IgM) antibody, and immunoglobulin G neutralizing antibody in immune monkey sera was enhanced to a much greater degree than was IgM antibody. There was a rapid decline in the complement requirement of IgM neutralizing antibodies over the course of immunization of the rhesus monkeys. V-Z neutralizing antibody titers in the presence of complement were higher than complement-fixing titers of the same sera in all groups of individuals studied. IgM neutralizing antibody for V-Z virus was demonstrable in all cases of varicella but in only 1 of 22 zoster cases, and V-Z IgM neutralizing antibody was not detectable in primary herpes simplex virus infections in which heterotypic antibody titer rises occurred to V-Z virus. Complement-fixing antibody for V-Z virus was absent in 19S serum fractions which contained IgM neutralizing antibody for the virus.     

Utility of direct immunofluorescence and virus culture for detection of varicella-zoster virus in skin lesions.

A direct immunofluorescence assay (DFA) with a monoclonal antibody from Ortho Diagnostic Systems was compared with conventional cell culture for the rapid detection of varicella-zoster virus (VZV) in 140 dermal lesions from 133 patients. A total of 79 (56%) specimens were positive for VZV: 40 (51%) by DFA alone, 2 (3%) by culture only, and 37 (47%) by both culture and DFA. After discordant analysis, the sensitivities and negative predictive values, respectively, were 97.5% (77 of 79) and 96.8% (61 of 63) for DFA and 49.4% (39 of 79) and 60.4% (61 of 101) for viral culture. Of the 39 positive viral cultures, VZV was isolated from 38 (97%) cultures in A549 cells, 23 (59%) in primary rhesus monkey kidney cells, and only 16 (41%) in MRC-5 cells. We conclude that DFA is the optimal method for rapid identification of VZV. In addition, better recovery of VZV in culture may be achieved by using A549 cells.     

Enzyme-linked immunosorbent assay for detection of antibody to varicella-zoster virus.

Primary varicella-zoster virus (VZV) infection is a serious illness in immunocompromised individuals, and a rapid, sensitive, and reliable assay to identify high-risk VZV-susceptible patients would be clinically useful. An enzyme-linked immunosorbent assay (ELISA) for antibody to VZV was compared with the fluorescent antibody-to-membrane antigen (FAMA) assay and found to be similar in both sensitivity and specificity. The antibody titers determined by both assays were also similar. The absence of antibody detected by ELISA correlated with susceptibility to VZV infection. Because it is simple to perform and has equivalent sensitivity to FAMA, ELISA should be useful for VZV antibody testing in diagnostic and research laboratories.     

Comparison of fluorescent-antibody-to-membrane-antigen test, indirect immunofluorescence assay, and a commercial enzyme-linked immunosorbent assay for determination of antibody to varicella-zoster virus.

The demand for sensitive and specific assays to determine immune status to varicella can be expected to increase with the anticipated availability of a varicella-zoster virus vaccine for use in nonimmune adults, especially health care personnel, and in immunosuppressed children. Although the fluorescent-antibody-to-membrane-antigen (FAMA) test remains the reference standard to which other tests are compared, simpler alternative assays are needed. In this study, the FAMA was compared with a simple indirect immunofluorescence assay (IFA) and a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to varicella-zoster virus. One hundred and twelve serum samples were screened by the FAMA test and IFA at a 1:5 dilution, and 100% agreement was found. Of these samples, 101 were available for testing by ELISA, and identical results were obtained with 97 samples (96% agreement). When the samples were screened at a 1:2 dilution, 99 of 101 results agreed. In addition, 31 spinal fluid samples were tested by all three methods. When screening was at a 1:2 dilution, there was 96.8% agreement between the FAMA test and IFA. When the cutoff value established for sera was used for the spinal fluid samples, there was 90.3% agreement between the ELISA and the FAMA test. Thus, both IFA and ELISA can be considered sensitive and specific alternatives to the FAMA test, and in addition, both use commercially available reagents.     

Comparison of three commercially available assays for detection of varicella-zoster virus antibody.

Three commercially available diagnostic assays for the detection of antibodies to varicella-zoster virus were evaluated to determine which would be the most suitable for our clinical laboratory. Three different methods were examined: an enzyme-linked immunosorbent assay (ELISA), latex agglutination (LA), and an indirect fluorescent-antibody technique. For the 141 serum specimens tested, the ELISA had an agreement of 90.1% and LA had an agreement of 92.2% with the indirect immunofluorescent-antibody technique. The ELISA had a lower sensitivity (85.6%) than LA (100.0%), but suffered from a low specificity (78.4%) compared with the ELISA (98.0%).     

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.     

Comparison of five assays for antibody to varicella-zoster virus and the fluorescent-antibody-to-membrane-antigen test.

Three commercially available assays (the Varicelisa Test Kit [Whittaker M.A. Bioproducts, Walkersville, Md.], the VZV Indirect Fluorescent-Antibody Test [Electro-Nucleonics, Inc., Columbia, Md.], and the Litton VZV Bio-EnzaBead Screen Kit [Litton Bionetics, Inc., Charleston, S.C.]) and two enzyme-linked immunosorbent assays used in our laboratory, one using a membrane-associated antigen and the other using a soluble antigen dotted on nitrocellulose paper, were compared with a varicella-zoster virus antibody reference assay, the fluorescent-antibody-to-membrane-antigen test. All of the assays compared favorably to the fluorescent-antibody-to-membrane-antigen test when evaluated for sensitivity (0.95), specificity (0.84), and test-retest reliability (79 to 96%), except for the Litton assay, which demonstrated significantly different results for all of the parameters tested (0.55, 1.0, and 69%, respectively).     

Use of murine monoclonal antibodies for laboratory diagnosis of varicella-zoster virus infection.

The laboratory diagnosis of varicella-zoster virus (VZV) infection was reevaluated by direct immunofluorescent-antibody staining (DFA) and centrifugation culture with newly available murine monoclonal antibodies. Specimen smears were examined by DFA using monoclonal antibodies to VZV and to herpes simplex virus types 1 and 2. Specimens were also inoculated into shell vials for centrifugation culture and into standard tube cell culture. Of 68 specimens tested from 60 patients, 39 (57%) were positive for VZV by at least one method. DFA was positive in 36 of 39 (92%); centrifugation culture was positive at 24 h in 23 of 39 (59%) and at 48 h in 31 of 39 (79%); and standard culture was positive in 25 of 39 (64%). Twenty-three of the 39 positive specimens (59%) were positive by all three techniques. Forty-three of the 60 patients were considered to have VZV by clinical criteria, and 35 of these 43 (81%) had laboratory confirmation of the diagnosis. These data confirm that DFA is the method of choice for the rapid laboratory confirmation of VZV infection. The centrifugation culture assay can provide an alternative method to DFA for the laboratory diagnosis of VZV infection.     

Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemagglutination techniques for detecting cytomegalovirus IgG antibody.

The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems. Cytomegalovirus (CMV) infection is an important cause of congenital brain damage and is also a major complication of both prolonged immunosuppressive therapy, especially in patients with organ transplants, and multi-donor blood transfusions. For serological diagnosis of infection, as well as for screening for antibody in patients and in blood donors, the solid-phase indirect radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques offer distinct improvements in sensitivity over previous methods. Although the principle of both tests, based on the detection of antigen-antibody reactions by means of a labelled anti-antibody, is the same, each possesses its own particular technical advantages and disadvantages, and both require their own expensive equipment for the reading of the results. There is still a lack of data on how they compare in sensitivity and specificity. The present study was undertaken to compare the two methods for the detection of CMV IgG and to evaluate them against the older techniques of complement-fixation (CF), passive haemagglutination (PHA) and anticomplement immunofluorescence (ACIF).     

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.     

Importance of recrudescent avian infection in West Nile virus overwintering: incomplete antibody neutralization of virus allows infrequent vector infection

After the acute infection period, birds persistently infected with West Nile virus (family Flaviviridae, genus Flavivirus, WNV) occasionally shed virus into the bloodstream, but these virions normally are inactivated by neutralizing antibody. The current work tested the hypothesis that these host neutralizing antibodies protect mosquito vectors from WNV infection and reevaluated the minimum WNV infectious dose necessary to infect Culex tarsalis Coquillett. To determine whether host antibodies protect mosquitoes from infection, Cx. tarsalis and Culex stigmatosoma Dyar were fed bloodmeals containing avian blood, WNV, and sera with or without WNV-specific neutralizing antibodies. When viral particles were completely bound by antibody, mosquitoes were protected from infection; however, when incompletely bound, WNV titers as low as 10(2.3) plaque-forming units (pfu)/ml resulted in 5% infection. These data indicated that avian antibodies were protective to mosquito vectors and were not dissociated during digestion. Because recrudescent viremias may not attain the same magnitude as initial acute viremias, Cx. tarsalis vector competence was reevaluated focusing on the fate of low-titered bloodmeals. Females were evaluated for vector competence after ingesting bloodmeals containing 10(2.2), 10(3.4), 10(4.5), 10(5.5), or 10(6.5) WNV pfu/ml. Infection increased with bloodmeal titer, with 1% of the mosquitoes ingesting 10(3.4) pfu/ml and 45% of the mosquitoes ingesting 10(6.5) pfu/ml developing disseminated infections. The incomplete neutralization of recrudescent virus may be sufficient to infect a low proportion of competent blood-feeding Culex mosquitoes and perhaps allow persistently infected birds to provide a mechanism for arbovirus overwintering.     

Evaluation of a direct immunofluorescence test for diagnosing gonorrhoea.

A new direct immunofluorescence reagent (Syva and Genetic Systems Inc) was evaluated for its ability to detect Neisseria gonorrhoeae in specimens from populations with a high prevalence of the infection. Gonorrhoea was diagnosed by culture in 45 of 105 (43%) urethral specimens from men and 17 of 90 (28%) urethral and 25 of 60 (42%) cervical specimens from women. In men the immunofluorescence test had a sensitivity of 84.4% and a specificity of 100%; Gram staining gave values of 94% and 100%, respectively. The sensitivity of the immunofluorescence test could be increased to 89% by testing duplicate smears. In women the immunofluorescence test had a sensitivity of 65% and a specificity of 98% for urethral samples and values of 72% and 94%, respectively for cervical samples. At both sites the sensitivity of the Gram stain was 40% and the specificity 100%. The testing of duplicate immunofluorescence smears increased the sensitivity to 76% for urethral and 88% for cervical samples.     

Experimental infection and immune response of guinea pigs with varicella-zoster virus.

An immune response (fluorescent antibody to membrane antigen) was detected in guinea pigs inoculated with varicella-zoster virus (VZV) adapted to guinea pig embryonic cells, including the Oka vaccine strain, even when inoculation was by an external route, i.e., nasal or corneal. Live or UV-inactivated virus having the same virus titer before irradiation was administered to guinea pigs by the corneal route, and antibody induction was detected only with live virus. The transmission of VZV from infected guinea pigs to noninfected ones was suggested by the appearance of antibody in the serum of the latter, who were kept in the same cage. The time course of the appearance of humoral and cellular immune responses in guinea pigs was examined by the fluorescent antibody to membrane antigen test and the skin reaction, with varicella antigen representing delayed-type hypersensitivity. When VZV was injected subcutaneously, skin reaction appeared as early as 4 days after inoculation, which preceded the appearance of detectable antibody by 2 to 6 days. In in vitro studies, the Oka vaccine showed a higher adsorption rate and better growth in guinea pig embryonic cells than did other wild-type strains when assayed by the infectious center assay. These results suggest that a system of VZV adapted to guinea pig cells and guinea pigs provides a good animal experimental model for immunological study of VZV infection.