Kleeman, J. N. MacCormack, S. J. Sasowski, and. E. E. Michaelson. 1980. Detection of Rocky Mountain spotted fever antibodies by a latex agglutination test. J.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1981, p. 214-216 0095-1 137/8t/010214-03$02.00/0
Vol. 13, No. 1
Detection of Typhus Antibodies by Latex Agglutination KARIM E. HECHEMY,' * JOSEPH V. OSTERMAN,2 CHRISTINE S. EISEMANN,2 L. BRUCE ELLIOTT,3 AND SANDRA J. SASOWSKI'
Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201'; Department of Rickettsial Diseases, Walter Reed Army Institute of Research, Washington, DC 200122; and Texas Department of Health, Austin, Texas 787563
A latex test for assay of antibodies to endemic and epidemic typhus rickettsiae is simple, group-specific, sensitive, and reproducible. Cross-reactivity within the typhus group was extensive.
We have developed a latex test for immunoassay of antibodies to Rickettsia typhi and to Rickettsia prowazekii. Erythrocyte-sensitizing substances from R. typhi and R. prowazekii were prepared by the method of Chang et al. (2) as described by Shirai et al. (9). Latex-R. typhi and latex-R. prowazekii suspensions were prepared by mixing (5) the reagents in the following order: latex (0.81 ,im; Difco Laboratories, Detroit, Mich.); erythrocyte-sensitizing substance in phosphate-buffered saline, pH 7.2; 0.1 M glycine-buffered saline, pH 8.1 (GBS); and GBS with 0.1% fatty acid-free bovine albumin (BAF; Sigma Chemical Co., St. Louis, Mo.). After each addition, the mixture was hand shaken for 2 min at room temperature. The volume of uncoated latex suspension was equal to the sum of the other components (latex, 0.5; antigen + GBS, 0.3; GBS-BAF, 0.2). For each antigen the range of optimum volumes of antigen used per 1.0 ml of latex-antigen (5), as determined by checkerboard titration, was 19 to 36 gl. Each 20-,ul test dose of latexantigen required between 0.38 and 0.72 jul of antigen. Thus, 1 ml of antigen was sufficient for 1,388 to 2,631 test doses. The reference test was the microimmunofluorescence (micro-IF) test as described by Philip et al. (7) with R. typhi and R. prowazekii antigens. The human sera used in this study were from the Walter Reed Army Institute of Research (WRAIR) and the Texas Department of Health. The WRAIR sera were from clinically documented typhus cases (obtained by J. J. Plorde, Bureau of Medicine and Surgery, Navy Department, Washington, D. C.) and were used in a previous study (9) for development of an indirect hemagglutination test for human antibody to typhus. Sera from Texas had been submitted for routine typhus testing. Whenever there was a discrepancy between the latex and micro-IF tests, clinical information was sought.
A total of 187 sera from 157 patients were tested. The latex-R. typhi test was evaluated with 175 sera from 149 patients and the latex-R. prowazekii test was evaluated with 12 sera from 8 patients. Three of these eight patients were diagnosed as having Brill-Zinsser disease (9). In addition, selected sera which were nonreactive to R. prowazekii and R. typhi by micro-IF were also tested by latex-R. prowazekii and R. typhi. They comprised 130 sera from patients with other pathological conditions (Table 3), including patients reactive by the nonspecific Weil-Felix test with titers -320. All sera were screened at a dilution of 1:16 in GBS-BAF, and nonreactive sera were assigned an arbitrary titer of 8 (10). Reactive sera were further titrated. The diluted serum (40 ,ul) and appropriate latex-antigen suspension (20 ttl) were applied to a glass slide, mixed, hand tilted for 6 min, and placed in a humidity chamber at room temperature for another 5 min. The titer was the reciprocal of the highest dilution showing definite agglutination. The threshold values of reactivity, indicative of the presence of specific antirickettsial antibody, were determined as for the latex-Rickettsia rickettsii test (5). These values were as follows: for a single specimen or an individual specimen of a pair, a titer >128 for micro-IF (5) and 264 for latex; for paired sera, a fourfold rise in titer to 264 for micro-IF and to -32 for latex. A titer one dilution below the minimum significant value was considered weakly reactive. Between-run precision was determined by assaying a reactive serum pool 17 times during 7 months with the same latex-antigen preparation. The modal titers were 512 (12 of 17 trials) for latex-R. typhi and 128 (9 of 17 trials) for latex-R. prowazekii. Reproducibiity within one dilution of the mode was 100% for latex-R. typhi and 94% for latex-R. prowazekii (16 of 17 trials). This indicates that the assay is highly precise and that the reagent is stable for at least 7 months.
214
VOL. 13, 1981
NOTES
The accuracy of a new immunoserological test, i.e., its ability to determine relative concentrations of antibody, can be determined by comparing the quantitative results of the new test to those of a reference test. Titers of the two tests can be compared directly if the threshold values of reactivity are the same. If not, the ratios of titers to the threshold values of reactivity can
be compared instead. Since the threshold values of reactivity were different for the latex and micro-IF tests, we compared the ratios by both tests with the respective antigen for 40 endemic typhus-reactive sera and 8 epidemic typhus sera (single specimens and second sera of pairs). The degree of correlation was evaluated statistically by the sign test (3). Of the 48 sera, 22 were ties. Of the nonties 14 had a higher ratio by the latex test and 12 had a higher ratio by the micro-IF. These nontie values did not fall in the critical region (18 trials). Thus, the accuracy of the latex test was not statistically different from that of the micro-IF test. Of the sera reactive by micro-IF, all 21 single sera and 19 of 21 pairs were also reactive by latex-R. typhi (Table 1). The remaining two serum pairs were from patients not diagnosed as having typhus; one of the two had rubella. Latex-R. prowazekii results were in agreement with micro-IF-R. prowazekii results for 12 sera from the eight patients diagnosed as having epidemic or recrudescent typhus (not included in Table 1). Because R. typhi and R. prowazekii are closely related antigenically (8), we examined the degree of cross-reactivity (Table 2) between them in each test with 25 clinically identified endemic typhus sera. Titers for the two antigens TABLE 1. Comparison of latex-R. typhi with microIF-R. typhi results' Micro-IF-R. typhi result Latex-R. typhi result
Reactive
Single Pairs Reactive Single Pairs
Weakly and nonreactive Single Pairs No. of patients
Weakly and nonreactive
Single Pairs
19
0
22 19
5
101 7
5
149
101 2b,
21
0
lb
21
21
No. of patients
102
a Number of single sera or number of pairs. Patients diagnosed as having epidemic or recrudescent typhus not included. Patients not diagnosed as having typhus. c One patient diagnosed as having rubella. b
215
TABLE 2. Cross-reactivity within the typhus group with endemic typhus sera No. of specimens Ratio of titers (R. typhi/R.
Test
prowazekii)
Total
Micro-IF Latex
25 25
1/2
1
2
4
8 1
15 13
2 7
0 4
TABLE 3. Latex-R. typhi and latex-R. prowazekii titers with selected sera nonreactive (
