Diagnosis of Postnatally Acquired Rubella by Use of Three Enzyme-

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1984, p /84/ $02.00/0 Copyright C 1984, American Society for Microbiology Vol. 20, No. 5 Diagnosis of Postnatally Acquired Rubella by Use of Three Enzyme- Linked Immunosorbent Assays for Specific Immunoglobulins G and M and Single Radial Hemolysis for Specific Immunoglobulin G PETER R. FIELD* AND CHUN MEI GONGt Virology Department, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia Received 29 May 1984/Accepted 14 August 1984 Three commercial indirect enzyme-linked immunosorbent assays (ELISAs) (RUBELISA, Enzygnost- Rubella, and Rubazyme) and a commercial single radial hemolysis (SRH) test (Rubazone) were evaluated for the diagnosis of acute rubella by testing 41 acute- (<7 days postonset) and convalescent-phase (8 to 82 days postonset) serum pairs from cases of rubella previously confirmed by signfficant change in the hemagglutination inhibition test titer. Specificity was tested by using 10 acute- and convalescent-phase serum samples from patients with rash not confirmed as rubella (control group). In testing for rubella-specific immunoglobulin G (IgG) antibody, Enzygnost-Rubella and Rubazyme confirmed infection in 40 and RUBELISA in 39 of the 41 proven rubella patients. For one patient all three ELISAs failed to show a significant titer rise. No false-positive diagnoses occurred in the control group, although a suspected infection was shown by Rubazyme in one patient. No specific IgM could be detected in this case. Single radial hemolysis confirmed infection in 39 of the 41 proven rubella patients, and one false-positive diagnosis occurred in the control group patients. Of the 43 convalescentphase serum samples, rubella-specific IgM was detected in 42 by Enzygnost-Rubella, in 41 by RUBELISA M, and in 39 by Rubazyme-M. For a rapid diagnosis with acute-phase sera, specific IgM detection by ELISA was most reliable in hemagglutination inhibition test-positive sera; of 18 such serum samples IgM was shown in 15 by Enzygnost-Rubella, in 13 by RUBELISA M, and in 11 by Rubazyme-M. Conversely, of 24 acute-phase serum samples with a hemagglutination inhibition test titer of <10, specific IgM was detected in only 5 by Enzygnost-Rubella, in 4 by RUBELISA M, and in 1 by Rubazyme-M. False-positive specific IgM results were shown by Rubazyme-M in two serum samples from one patient in the control group. These serum samples were negative with the other two ELISA methods. Since 1967 diagnosis of rubella infection has been carried out mainly by serological means by the hemagglutination inhibition test (HAI). This has been done by demonstrating fourfold or greater rises in HAI titers between paired serum specimens; however, in cases where an acute-phase blood specimen is not available, and it is not possible to demonstrate a rise in antibody titer, specific immunoglobulin M (IgM) antibody testing has been done by separating the IgM fraction of serum from IgG and testing this fraction for rubella HAI antibody activity (24). The isolation of rubella virus is seldom used, as a diagnosis of rubella is obtained more readily by serological testing (12). During recent years several other techniques have been described for the measurement of rubella IgG or IgM antibody, or both. Antibody measured by passive hemagglutination (3) is not of the IgM class and shows a considerably delayed immune response and, owing to the urgency of rapid diagnosis of rubella infection in pregnant women, demonstration of seroconversion by this technique is impractical. Single radial haemolysis (SRH) (21) provides a simple means for confirmation of recent rubella by showing an increase in antibody level in paired serum specimens, but it cannot be used to detect IgM antibody (13). On the other hand, enzyme-linked immunosorbent assay (ELISA) (23) and solid-phase radioimmunoassay (16) have been shown to detect significant IgG antibody rises in paired serum specimens from cases of rubella. They can also measure specific IgM antibodies in nonfractioned sera. * Corresponding author. t Present address: institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming, People's Republic of China. 951 These immunoassays have advantages over HAI, passive hemagglutination, and SRH in that they have the potential for automation, and precise numerical read-out replaces visual interpretation. However, solid-phase radioimmunoassay suffers from the disadvantages of short shelf life, high cost of reagents and counters, and possible safety hazards. For these reasons the ELISA technique is the one which is being increasingly developed, evaluated, and used in viral serology. Other studies have shown ELISA (4, 10, 20, 23, 25) to have precision similar to that of HAI in determination of immune status and diagnosis of recent infection. However, to our knowledge no fully documented comparative study evaluating commercial ELISA systems for the diagnosis of recent acquired rubella infection has been published. Similarly, other studies using "in-house" laboratory-prepared reagents have shown the suitability of SRH for diagnosing recent infection (4, 11, 13, 19, 21), but only brief mention has been made of the use of commercial reagents for such purpose (22). This communication describes the evaluation of three commercial indirect ELISA systems for IgG, three commercial indirect ELISA systems for IgM, and one commercial SRH system and compares them with an in-house HAI for the diagnosis of postnatally acquired rubella. Commercial SRH tests are not widely available, due to their short shelf life. All the ELISA systems and the SRH used a single dilution of serum. Besides evaluating the measurement of rubella IgG by ELISA and SRH for demonstrating significant antibody rises in cases of recent rubella, rubella IgM ELISA testing was carried out on each serum pair for the following reasons: (i) to determine whether acute-phase serum specimens could be used to obtain an early diagnosis,

2 952 FIELD AND GONG J. CLIN. MICROBIOL. (ii) to obtain an indication of the persistence of rubella IgM as measured by ELISA so that its role in retrospective diagnosis could be more accurately defined, (iii) to confirm that the rising titers are evidence of primary rubella infection, and (iv) to resolve any discrepancies occurring in serum pairs between HAI tests and ELISA rubella IgG results. MATERIALS AND METHODS Clinical specimens. The study material consisted of a total of 105 serum specimens. The age of the patients ranged from 5 to 40 years, with the great majority being adults. These serum specimnens were divided into two groups. (i) Confirmed rubella infection group. Paired serum specimens were collected from 41 patients during the acute and convalescent stages of rubella infection (patients 1 through 41). These cases were confirmed as rubella by demonstration of seroconversion or significant rise in antibody titer as determined by HAI. In two cases three additional serum specimens were collected, so that a total of 85 specimens was tested. It was not possible to determine the exact date of onset of symptoms for some patients, but in all cases the collection of the acute-phase serum specimens was within 1 week of onset. Convalescent-phase serum specimens were collected at periods ranging from 8 to 82 days after onset, but mainly in the second and third weeks after onset. (ii) Control group. Paired serum specimens were collected from 10 patients during the acute and convalescent stages of an illness resembling rubella (patients 42 through 51). None of these cases could be shown to be due to rubella infec,tion, as all patients had stationary rubella HAI titers. Two cases were confirmed by seroconversion as measles, two as adenovirus infections, and one as dengue fever. The remaining five cases with stationary rubella titers could not be associated with any other etiological agent, and tests for rubellaspecific IgM in the three cases with HAI antibody were negative. HAI. A microtiter technique was used (9) with trypsinized human 0 cells at a concentration of 0.3% with HEPES (N-2- TABLE 1. hydroxyethylpiperazine-n'-2-ethanesulfonic acid) buffer (ph 6.2) containing 0.5% bovine albumin fraction V as diluent and 4 U of commercially available rubella antigen. Sera were pretreated with heparin-manganous chloride before testing. ELISA for rubella IgG. Enzyme immunoassays were conducted on all serum specimens by three indirect ELISA systems for IgG with commercial reagents. The reagents used were RUBELISA (M. A. Bioproducts, Walkersville, Md.), Enzygnost-Rubella (Calbiochem-Behring, La Jolla, Calif.), Rubazyme (Abbott Laboratories, North Chicago, Ill.). All the test systems used the principle of indirect ELISA, and the procedure followed was that recommended by the manufacturer, with the exception of one modification in the Enzygnost-Rubella system. This involved the stopping of the enzyme reaction after a shorter time (usually 10 to 15 mins) than that recommended by the manufacturer (45 min). When the reaction was stopped after 45 min, background (control antigen) absorbance values rose considerably, resulting in many false-negative results. A comparison of the principle characteristics of the three ELISA test systems and SRH is shown in Table 1. The solid-phase component of each system consisted of antigen attached to either the well in a plastic microplate (RUBELISA, Enzygnost-Rubella) or a bead (Rubazyme). With the exception of Rubazyme, all systems employed a control antigen to test for nonspecific reactivity. All systems used an anti-human IgG conjugate. The composition of serum and conjugate diluents, as well as washing solutions, could not be obtained from the manufacturer, with the exception of RUBELISA, which has a washing solution of phosphate-buffered saline plus 0.05% Tween and Rubazyme, which uses water for washing. For RUBELISA and Enzygnost-Rubella, washing was performed with a Dynatech Miniwash, and absorbances of the solutions in each well were measured directly in the plate with a vertically measuring photometer (Dynatech MR580 MicroELISA Auto Read- Comparison of test procedure for three indirect ELISA systems and SRH for rubella IgG for diagnosis of recent rubella infection ELISA Characteristic Enzygnost- SRH RUBELISA Rubella Rubazyme (Rubazone) (Calbiochem-Behring) Solid-phase antigen Microplate in strips Microplate Bead NAa Control antigen Yes Yes No Yes Serum dilution 1/25 1/200 1/20 Undiluted Serum incubation 0.75 h at 20 to 25 C 2 h at 20 to 25 C 1 h at 37 C 20 h Enzyme/substrate Alkaline phosphate/pnppb Alkaline phos- Peroxidase/OPDC NA phate/pnpp Conjugate incubation 0.75 h, RTd 1 h, 370C 1 h, 370C NA Substrate incubation 0.75 h, RT 0.75 h, RTe 0.5 h, RT NA Absorbance reading 405 nm 405 nm 492 nm NA Time 3.25 h per 48 tests 3.75 h per 48 tests 5 h per 100 tests 20 h per 60 tests Approx. costf of reagents per $4.30Y $2.10 $3.309 $1.60 to $2.00 test serum Stability of reagents >1 yr >1 yr >1 yr 3 weeks Evaluation of paired sera Absorbance ratio (.1.65) HAI ratio (.4x) Absorbance ratio IUh (HAI) ratio a NA, Not applicable. b pnpp, p-nitrophenyl phosphate. c OPD, o-phenylenediamine-2-hydrochloride. d RT, Room temperature (20 to 25 C). ein practice, 10 to 15 min was found to be the optimal time. f Costs are shown in Australian dollars. 8 Based on cost of duplicate tests as recommended by manufacturer. h IU, International units. (.1.65) (24x)

3 VOL. 20, 1984 DIAGNOSIS OF POSTNATALLY ACQUIRED RUBELLA 953 TABLE 2. Comparison of test procedure for three indirect ELISA systems for rubella IgM Characteristic RUBELISA M Enzygnost-Rubella (Behringwerke) Rubazyme-M Solid-phase antigen Microplate in strips Microplate Bead Control antigen Yes Yes No Serum pretreatment" Yes No Yes Serum dilution 1/45 1/40 1/200 Serum incubation 2 h at 37 C 1 h at RTh 2.5 h at 45 C Enzyme/substrate Alkaline phosphate/pnpp' Alkaline phosphatase/pnpp Peroxidase/OPD" Conjugate incubation 2 h at RT 1 h at 37 C 1.5 h at 45 C Substrate incubation 0.75 h at RT 0.75 h at RT 0.5 h at RT Absorbance reading 405 nm 405 nm 492 nm RF false-positive results No Yes No Time 6 h per 48 tests 3.5 h per 48 tests 6 h per 50 tests Approx. cost of reagents per $9.50 $2.30 $6.90' test serum Stability of reagents >1 yr >1 yr >1 yr Interpretation of results Absorbance values of as positive Absorbance values of.0.2 as posi- Rubazyme-M index tive >1.09 as positive C To remove the interfering effect of IgG or RF. RT. Room temperature (20 to 25'C). pnpp. p-nitrophenyl phosphate. d OPD. o-phenylenediamine-2-hydrochloride. ' Based on cost of duplicate tests as recommended by the manufacturer. Costs are shown in Australian dollars. er; Dynatech Laboratories, Inc., Alexandria, Va.). For Rubazyme, washing was done with a Pentawash 1I (Abbott Laboratories) and absorbances were measured in a Quantum II spectrophotometer (Abbott Laboratories). The methods for evaluating the results for paired serum samples for the presence of a significant increase in rubella IgG appear in detail as footnotes to Table 4. SRH test. All sera were tested with commercial reagents (Rubazone; Scientific Measuring Instruments, Sydney, Australia). Serum samples (5 jl), inactivated at 56 C, were added to wells in a system that was similar to that described by Vaananen and Vaheri (21), except that sheep erythrocytes were used and complement was added to the plates after overnight radial diffusion of IgG antibody. To minimize interlaboratory variability, a standard curve was produced by using calibrators standardized against the World Health Organization 1,000-IU standard for anti-rubella serum, and all SRH titers were expressed in international units. Fourfold increases in antibody level indicated suspected current infection. ELISA for rubella IgM. All serum specimens were tested for rubella IgM antibody by three indirect ELISA systems with commercial reagents. The reagents used were RU- BELISA M, Enzygnost-Rubella (Behringwerke, Marburg, West Germany), and Rubazyme-M. All the test systems used the principal of indirect ELISA. and the procedure used was that recommended by the manufacturer. The shortened enzyme action time, which was found necessary in the Enzygnost-Rubella IgG ELISA, was not required in the IgM ELISA. The principal differences in methods among the three tests systems are shown in Table 2. The solid-phase component of each system, the method of washing, and the absorbance reading were similar to that described for the IgG ELISA. All systems used an antihuman IgM conjugate. The composition of serum pretreatment mixtures and serum and conjugate diluents could not be obtained from the manufacturers. To overcome the interfering problem of IgG and rheumatoid factor (RF), an initial serum pretreatment step was used in RUBELISA M to remove IgG. In Rubazyme-M the serum incubation buffer is said to neutralize high concentrations of serum RF (Martin Armstrong, Abbott Laboratories, personal communication). With Enzygnost-Rubella there was no serum pretreatment to remove IgG or RF, and in cases of positively reacting sera it was necessary to repeat the IgM determination after removal of possible RF by a method such as absorption with IgG-coated latex particles (15). (Behringwerke now recommends pretreatment of sera with a new reagent, RF-Absorbent, for removing RF. At the time this study was conducted this new reagent was unavailable in Australia.) The World Health Organization recommends that all viralspecific IgM determinations should be made on a purified IgM fraction (1). The calculation and interpretation of the results varied for the three methods and are shown as footnotes to Table 4. Discrepant and equivocal results. All patient serum specimens showing discrepant results were repeat tested to show that consistent results were obtained. In addition, equivocal TABLE 3. Comparison of ELISA and SRH for 41 acute and convalescent-phase serum pairs from cases of rubella confirmed by significant change in HAI titer and 10 acute- and convalescent-phase serum pairs from cases with rash not confirmed as rubella (control group) for diagnosis of acute rubella by demonstration of significant rise in antibody level" ELISA No. of SRH (Rubazone) Group sr p RUBELISA Enzygnost-Rubella Rubazyme serum pairs Positive Negative Positive Negative Positive Negative Positive Negative Confirmed rubella (95.1) 2 (4.9) 39 (95.1) 2b (4.9) 40 (97.6) 1" (2.4) 40 (97.6) 16 (2.4) Control group 10 1 (10) 9 (90) 0 10 (100) 0 10 (100) 0 10' (100) "Values represent the number of serum pairs (%). Patient 12 gave results indicating suspected infection. Patient 47 gave results indicating suspected infection.

4 954 FIELD AND GONG J. CLIN. MICROBIOL. results were retested in duplicate. The SRH results from patient 42 were confirmed in an independent laboratory. RESULTS Detection of a significant rise in rubella IgG antibody levels in paired serum specimens determined by the three ELISA methods and SRH. The ability of the three ELISA methods and SRH to detect significant rises in rubella IgG antibody between acute- and convalescent-phase sera from 41 cases of clinical rubella confirmed by significant HAI titer rises is summarized in Table 3. Also included are the results obtained by testing 10 serum pairs from patients with rash not confirmed as rubella (control group). Table 4 shows the actual test results and time of collection of specimens for sera in test and control groups which gave discrepant results. (The data on patient serum specimens not shown are available from the authors upon request.) SRH failed to confirm infections in two patients (patients 3 and 12). RUBELISA detected significant changes in 39 serum pairs. It failed to show any change in antibody level in patient 3 but gave results indicating a suspected infection in patient 12. Enzygnost-Rubella and Rubazyme detected significant antibody rises in all cases, except for patient 12 (Table 4), in whom the results from these two methods indicated a suspected but not confirmed infection. Patient 12 was not hypogammaglobulinemic. With the control group, SRH showed a seroconversion in one patient (patient 42). This patient also showed seroconversion by HAI to dengue fever virus, and the convalescent serum contained dengue fever virus IgM. However no rubella IgM antibody was demonstrated in either the acuteor convalescent-phase serum (Table 4). None of the three ELISA techniques gave positive results in the control group, except for Rubazyme, which in one case (patient 47) indicated a suspected infection. Although the Diagnostic Ratio was.1.65, the Rubazyme index was <1.00 for both serum specimens. Comparison of the three ELISA methods for detection of rubella IgM antibody in acute- and convalescent-phase serum specimens. To determine whether acute-phase serum specimens could be used to obtain an early and hence rapid diagnosis, the 42 acute-phase serum specimens (collected <7 days after onset) were compared with the 43 convalescentphase serum specimens (collected.7 days after onset) obtained from the 41 cases of confirmed rubella infection (Table 5). Of the 42 acute-phase serum specimens, 17 (40.5%) were positive by RUBELISA M, 20 (47.6%) were positive by Enzygnost-Rubella, and 12 (28.6%) were positive by Rubazyme-M. Of the 43 convalescent-phase serum specimens (collected between 7 and 82 days after the onset of infection), 41 (95.4%) were positive by RUBELISA M, 42 (97.7%) were positive by Enzygnost-Rubella, and 39 (90.7%) were positive by Rubazyme-M. Details about specimens which gave discrepant results are shown in Table 4. Rubella-specific IgM could be demonstrated in only 6 of TABLE 4. Patients with discrepant results in a comparison of three ELISA systems for rubella IgG and IgM with SRH for the diagnosis of acquired rubella in 85 serum specimens from 41 cases of rubella confirmed by significant change in HAI titer (patients 1 through 41) and 10 acute- and convalescent-phase serum pairs from 10 cases with rash other than rubella (patients 42 through 51). IgG IgM Days RUBELISA Enzyg- Rubazymed Patient no. after HAI SRHa Gb nost' RUBELISA Rubazymeonset Ab Critical Estimated Rubazyme Diag. Me Enzygnostr ml s ratio HAI index Ratio Proven rubella infection group 1 1 <10 < < ' < < < '0.80 _ < _ < _ ' <10 < < _ < < '0.80 _ <10 < < < ' < < < < < ' < < ' <10 < < <10 < < _0.80 _ <10 < < '-0.80 ' Continued on following page

5 VOL. 20, 1984 DIAGNOSIS OF POSTNATALLY ACQUIRED RUBELLA 955 TABLE 4-Continiued IgG Days RUBELISA Enzyg- Rubazyme" Patient no. after HAl SRH" Gb nost RUBELISA Rubazymeonset Ab Critical Estimated Rubazyme Diag. Me Mq Abs. ratio HAI index Ratio IgM 30 1 <10 < < ' _0.80 ' <7 <10 < < _ <10 < < ' < _ _ <10 < < ' > < < _ < < ' NTh NT NT NT _ NT NT NT NT _ < ,560 ' <10 < < <7 20 < _ _ _ < < <10 < < Patients with rash other than rubella (control group) < ' ' < < < < a SRH results are expressed in international units. Values of.8 indicate evidence of previous infection. RUBELISA G absorbance values of.0.17 (obtained from a calibration curve) are equivalent to an HAI titer of.1:8. The critical ratio is the ratio of the mean RUBELISA value of the convalescent-phase serum to that of the acute-phase serum. A critical ratio of '1.65 indicates a highly significant increase in antibody titer, and a ratio of 1.47 to 1.64 is comparable to a four-fold increase in HAI titer. Abs., Absorbance values. Enzygnost-Rubella also involves the construction of a calibration curve, but the test ELISA values are converted back to an estimated HAI titer. d Rubazyme absorbance values are expressed as the Rubazyme index. This is the ratio of the mean specimen absorbance to the mean of duplicate low-positive control absorbance values. An index of _1.00 is positive for rubella IgG antibody. The Diagnostic Ratio is the ratio of the Rubazyme index of the convalescentphase serum to that of the acute-phase serum. A ratio of '1.65 is confirmation of recent rubella infection. e RUBELISA M values of.0.28 (from a calibration curve) are positive. Values of '0.19 are negative, and intermediate values are equivocal. f Enzygnost-Rubella values are expressed as the net absorbance. Values of.0.2 are positive. 8 Rubazyme-M values are expressed as an index by dividing the mean duplicate test specimen absorbances by the mean of the triplicate low-positive control values. A Rubazyme-M index of.1.09 is positive and an index of '0.91 is negative. Intermediate values are equivocal. h NT, Not tested. the 24 (26.1%) acute-phase serum specimens with an HAI titer of <10 by any of the three ELISA methods. Of these six acute-phase specimens, rubella IgM was detected by all three ELISA methods in one case (patient 1). RUBELISA M and Enzygnost-Rubella were positive in two cases (patients 6 and 19). Enzygnost-Rubella was the only test positive in two cases (patient 22 and 29), and RUBELISA M was the only test positive in one case (patient 31). In summary, of the 24 HAI-negative acute-phase serum specimens, specific IgM was detected in only 5 (20.7%) by Enzygnost-Rubella, 4 (16.7%) by RUBELISA M, and 1 (4.2%) by Rubazyme-M. On the other hand, specific IgM was detected in 10 (55.6%) of 18 acute-phase serum specimens with HAI titers of.10 by all three ELISA methods. Of the remaining eight acute-phase serum specimens, RUBELISA M and Enzygnost-Rubella were positive in two (patients 4 and 18). Enzygnost-Rubella and Rubazyme-M were positive in one alone (patient 40), RUBELISA M was positive in one alone (patient 5), and Enzygnost-Rubella was positive in two alone (patients 14 and 35). Two serum specimens were negative by all three methods (patients 10 and 37). In summary, of the 18 HAI-positive acute-phase serum specimens, Enzygnost-Rubella was positive in 15 (83%), RUBELISA M was positive in 13 (72.2%), and Rubazyme-M was positive in 11 (61.1%).

6 956 FIELD AND GONG J. CLIN. MICROBIOL. TABLE 5. Comparison of the three rubella-specific IgM ELISA methods for the 85 serum samples from the confirmed rubella infection group (42 acute-phase serum specimens collected <7 days after onset and 43 convalescent-phase serum specimens) and the 20 serum specimens from the control group (rash not confirmed as rubella)' GroupNo. of serum RUBELISA M Enzygnost-Rubella Rubazyme-M specimens Positive Negative Positive Negative Positive Negative Confirmed rubella 42 (acute) 17 (40.5) 25b (59.5) 20 (47.6) 22 (52.4) 12 (28.6) 30' (71.4) 43 (convalescent) 41 (95.4) 2d (4.6) 42 (97.7) 1 (2.3) 39 (90.7) 4 (9.3) Control (100) 0 20 (100) 2 (10) 18 (90) a Values represent the number of serum specimens (%). b Three specimens gave equivocal results. c Two specimens gave equivocal results. d One specimen gave equivocal results. The overall detection rate of rubella IgM antibody by using the three ELISA methods with the 85 serum specimens from the confirmed rubella infection group (acute- and convalescent-phase specimens combined) was 58 (68.2%) for RUBELISA M, 62 (72.9%) for Enzygnost-Rubella, and 51 (60%) for Rubazyme-M. Equivocal results were obtained with four serum specimens tested by RUBELISA M (patients 20, 32, 40, and 41) and two serum specimens tested by Rubazyme-M (patients 4 and 31). In the group with rash not confirmed as rubella (control group) 2 of 20 serum specimens (10%) were positive by Rubazyme-M (Table 5). These two specimens were acuteand convalescent-phase sera from patient 44. No falsepositive results were obtained with either RUBELISA M or Enzygnost-Rubella. Persistence of rubella-specific IgM. Enzygnost-Rubella detected rubella-specific IgM in all sera collected between 7 and 82 days. RUBELISA M detected specific IgM in all such sera up to 63 days after onset (patient 40). At 82 days (patient 41) this test gave an equivocal result which was just below the positive cut-off point (Table 4). Rubazyme-M was reliable in detecting rubella-specific IgM up to 26 days after onset (patient 33) but detected this antibody irregularly thereafter. For example, Rubazyme-M was negative at 28 days (patient 34), 29 days (patient 36), and 63 days (patient 40), but on the other hand, it detected rubella IgM in a different patient (patient 35) at 28 days. It also was positive at 30 days (patient 37), 35 days (patient 38), 36 days (patient 39), and 82 days (patient 41). It should be pointed out that the serum collected from patient 17 at 15 days after onset was an exception to the above in that none of the three ELISA systems detected rubella IgM, despite the fact that significant rises in antibody level were shown in this patient by HAI, SRH, RUBELISA, Enzygnost-Rubella, and Rubazyme. There was insufficient serum remaining to test for rubella IgM by sucrose density gradient ultracentrifugation. Thus, all of the 41 patients with HAI rising titers were confirmed as having rubella by specific IgM testing, with the sole exception of patient 17. DISCUSSION Both the ELISA and SRH have the advantages over HAI in that nonspecific inhibitors of hemagglutination which may cause false-positive results do not need to be removed before testing (14), results can be read objectively, and quantitation of antibody levels can be estimated on single dilutions of serum. In comparison to conventional truncated HAI serum dilution titers, where reproducibility is acceptable so long as replicate titers remain within a twofold range, ELISA test results are more accurate because they are free to take on any of a continuum of values, usually with a reproducibility of ±10% (coefficient of variation) (5). RUBELISA and Rubazyme use the continuous nature of ELISA values, whereas Enzygnost-Rubella IgG results have to be converted back to an HAI titer, with the inherent disadvantage that only antibody increases of fourfold or greater are significant. Enzygnost-Rubella IgM uses a predetermined fixed cut-off absorbance level, which does not deal effectively with the problem of interassay variation. There is no general agreement as to what method of reporting of test results is preferable in ELISA (5). Other investigators (4, 20, 23, 25) have found that enzyme immunoassay detects rising levels of rubella IgG antibody later in the course of infection than the HAI test, and this may in fact be an advantage in the diagnosis of some infections where the first serum specimen is drawn late and a rise in HAI antibodies cannot be shown. However, this was not seen in the present study, as acute-phase serum specimens were collected early in the first week after onset of infection. As SRH detects only IgG antibody, the advantages in relation to the timing of collection of specimens are the same (21) as for IgG ELISA. Rubella-specific IgM was detected in 29 to 48% of acutephase serum specimens from confirmed rubella cases, depending on the ELISA method used, and thus there appears to be a limit to the use of these tests for rapid diagnosis on single acute-phase serum specimens. However, the acutephase serum specimens tested in this study were collected within 2 to 3 days of the onset of a rash, and this may explain why higher detection rates of specific IgM were not obtained. Other reports have also indicated that rapid diagnosis by estimating specific IgM in acute-phase serum specimens is limited. For example, Coxiella burnetii-specific IgM was detected by ELISA in only 4% of the acute-phase serum specimens collected during the first week after onset of Q fever infection and in 38% of the serum specimens collected during the second week (7). In another study (C. M. Gong, D. W. T. Ho, P. R. Field, and A. M. Murphy, J. Virol. Methods, in press) to evaluate the use of specific IgM by ELISA for the diagnosis of echovirus type 11 infections, echovirus type 11-specific IgM was detected in only 58% of the acute-phase serum specimens. Using an ELISA for mumps-specific IgM, Meurman et al. (17) detected mumpsspecific IgM in 96% of the first available serum specimens from clinically typical mumps infection. However, in their report, the time of collection after onset was not stated, and it appears that many of the first serum specimens were collected during convalescence, as rising titers by complement fixation tests were obtained in only 71% of these cases. However, in some viral infections, such as hepatitis A, which have long incubation periods, specific IgM is detectable at the time of onset. It is difficult to explain the failure to detect rubella-specific IgM by all three methods in one case (patient 17), and there

7 VOL. 20, 1984 DIAGNOSIS OF POSTNATALLY ACQUIRED RUBELLA 957 was no other evidence that this patient was undergoing a secondary infection. The possibility of hypogammaglobulinemia was ruled out, as total immunoglobulin levels were shown to be normal. Alternatively, the IgM response may have been of very short duration. In the control group, the two false-positive results detected by Rubazyme-M are of concern. It has been shown that Rubazyme-M can produce false-positive results due to the presence of heterophil antibodies (18), but the manufacturer claims that false-positive results only occurred in early Rubazyme-M kits and not in subsequently modified kits. The kits used in the present study were of the latter type. Despite continuous improvements being made, a later study (2) has shown that false-positive results can still occur in Rubazyme-M with sera without heterophil antibodies. A Rubazyme-M confirmatory neutralization test was not available in Australia at the time of our studies. In the absence of a reliable confirmatory test or the incorporation into the test of a control antigen bead to test for nonspecific reactivity, all positive results obtained by Rubazyme-M should be confirmed by another method. Commercial kits and reagent sets are now being increasingly promoted for use in microbiology, especially for serological diagnosis of viral infections, but unlike in America, where the U.S. Department of Health, Education and Welfare has granted authority to the Food and Drug Administration to regulate this expanding field, there is no control of the sale and use of such products in Australia and certain other countries. Thus, laboratory workers in these countries have no idea as to the accuracy and precision of a given kit apart from what is provided by unsolicited, independent studies, such as the present report, or the performance characteristics described as a result of the evaluation of the manufacturer. Although no bias can be directly ascribed to the in-house testing of the manufacturer, it would seem to be desirable for countries lacking regulatory approval to have some authoritative control over the use of commercial diagnostic reagents in their countries. This could perhaps be accomplished by the testing of new commercial diagnostic reagents and systems in large government laboratories where the necessary clinical material and scientific and technical expertise are available for such evaluations. This would guard against products or batches of products not approved by the Food and Drug Administration which are either unsuitable or lacking in diagnostic precision being used in other countries. In many kits the identity of some of the reagents is not revealed, and this applies to the test systems evaluated in this communication. It has been suggested that no clinical chemist should ever agree to employ secret reagents (6). It is appreciated that companies expend much effort in producing reagent systems in competition with rival manufacturers and that, accordingly, they may not wish to make known some details of such systems. However, until more detailed information on the ingredients used is available from manufacturers, many reputable microbiologists will be reluctant to use such commercial reagent sets. For the diagnosis of rubella when acute- and convalescentphase serum pairs are available, two commercial rubella IgG ELISA systems, Enzygnost-Rubella and Rubazyme, appeared to be suitable alternatives to the more laborious, yet well-proven and understood, HAI test. RUBELISA G did not detect infection in one patient. The commercial SRH, Rubazone, although the simplest test to perform, was the least satisfactory, as it failed to detect seroconversion in two patients and gave a false-positive result in another patient. Of the three commercial ELISA systems tested for rubella-specific IgM, Enzygnost-Rubella and RUBELISA M appeared to be less costly yet comparable alternatives to the cumbersome, time-consuming, and relatively expensive reference technique, HAI testing on IgM fractions obtained after sucrose density gradient ultracentrifugation (8). Rubazyme-M was less satisfactory in our hands. It was less sensitive than the other two systems, and false-positive results occurred. For a rapid diagnosis, acute-phase sera with low levels of HAI antibodies can be tested by Enzygnost-Rubella or RUBELISA M provided it is recognized that this is only a screening test. If equivocal or negative results are obtained with an acute-phase serum specimen, a second early-convalescent specimen should be tested. It is essential that all available evidence (all serological results, previous test results, and as much accurate clinical data as possible) be taken into consideration before making a diagnosis of rubella when this occurs in pregnancy. When the result is difficult to interpret or results are not consistent with the history, we would recommend that the tests be repeated and that sera giving equivocal results should be tested in a different assay. ACKNOWLEDGMENTS We are indebted to all our colleagues in the Virology Department for their whole-hearted assistance and A. M. Murphy for his advice in preparation of the manuscript. 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