Development and Evaluation of Dot-Immunobinding Assay for Detection of Scrub Typhus Infection in Leptotrombidium fletcheri (Acari: Trombiculidae) T. M. HO, SITI SHARA, A. S. KOAY, AND Y. M. CHEONG Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia

J. Med. Entomol. 29(4): 611-613 (1992)

KEY WORDS Acarina, dot-immunobinding assay, direct fluorescent antibody technique, Leptotrombidium fletcheri

SCRUB TYPHUS IS A DISEASE caused by the organism Rickettsia tsutsugamushi. The disease is endemic in the Asian-Pacific region (Oaks et al. 1983). Transmission of scrub typhus is through the bite of infected larvae of certain species of trombiculid mites belonging to the genus and subgenus Leptotrombidium; these larvae also are termed "chiggers." There are three vector species in Malaysia: L. deliense (Walch), L. fletcheri (Womersley & Heaslip), and L. arenicola Traub. For a chigger to be a vector of scrub typhus, there must be transovarial and transtadial transmission of rickettsiae in the mite. These two modes of transmission occur in naturally infected colonies of L. fletcheri and L. arenicola. It was not possible, however, to demonstrate transovarial transmission in populations of these two species that were not naturally infected (Robinson et al. 1976, Shirai et al. 1979). Because of the existence of these two different populations in the same species, detection of infection in a resident chigger population is important for determining the risk of scrub typhus infection in an area. Traditional methods for the detection of scrub typhus infection in chiggers are isolation in susceptible hosts and direct fluorescent antibody technique (DFAT). Isolation in animals is tedious and time consuming. The DFAT is comparatively rapid; however, it requires a fluorescent microscope and is thus, at present, not practical for field application. The DFAT also requires experienced personnel to read the results. One relatively simple, accurate, and low-cost technique which has potential as a field tech-

nique for detecting scrub typhus in naturally infected chiggers is the dot-immunobinding assay (DIBA). The DIBA was first developed by Hawkes et al. (1982) and has been used in the detection of a number of organisms (Tsai et al. 1989, Oprandy & Long 1990, Woods & Iwen 1990). The objectives of this study were to adapt the DIBA for detecting scrub typhus infection in L. fletcheri and to compare the specificity and sensitivity of DIBA with DFAT. Materials and Methods Chiggers. The chiggers used in this study were from infected and noninfected L. fletcheri colonies kept in the Institute for Medical Research, Kuala Lumpur. These colonies had been maintained for >50 generations in the laboratory. Infections in the chiggers were confirmed first by isolation in white mice and subsequently were monitored by DFAT for each successive generation. Rapmund et al. (1969) reported that an infected female L. fletcheri gave rise to 100% infected chiggers; thus, it was assumed that all the chiggers in the infected lines were infected with R. tsutsugamushi. Unfed chiggers from each infected and noninfected population were assigned randomly to either the DFAT or DIBA in a single-blind study. Hyperimmune Serum. New Zealand white rabbits were immunized intravenously with 0.5 ml of yolk sac suspension of the Karp strain of R. tsutsugamushi; the suspension contained =106-107 organisms per milliliter. Two weeks later, the rabbits were boosted intravenously

0022-2585/92/0611-0613$02.00/0 © 1992 Entomological Society of America

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ABSTRACT A dot-immunobinding assay (DIBA) was compared with a direct fluorescent antibody technique (DFAT) for the detection of Rickettsia tsutsugamushi infection in Leptotrombidium fletcheri (Womersley & Heaslip). Laboratory colonies of infected and noninfected chiggers were examined. The relative proportions of positive, negative, and indeterminate results were significantly different between DIBA and DFAT for infected but not for noninfected chiggers. DIBA was more sensitive and had a better negative predictive value and a lower false negative percentage than DFAT. It was concluded that DIBA is a suitable alternative to DFAT for detecting scrub typhus infection in chiggers.

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JOURNAL OF MEDICAL ENTOMOLOGY

Table 1. Detection of scrub typhus infection in L. fletcheri by direct fluorescent antibody technique (DFAT) and dot-immunobinding assay (DIBA) Chigger population

Total no. examined

Infected Noninfected Total

200 200 400

+

DFATa -

+/-

+

73 5 78

20 91 111

7 4 11

92 6 98

DIBA" 5 91 96

+/3 3 6

+ , positive; —, negative; +/—, indeterminate.

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with another 0.5 ml of that suspension. Serum jugate (Cappel) were added. Each disk was was obtained 1 and 2 wk later, and antibody incubated for 2 h at 37°C. The conjugate was titers were determined by indirect fluorescent removed, and each disk was washed with TBS. antibody technique (Bozeman & Elisberg 1963). 150 fi\ of a substrate solution containing 5 mg If the titer was >l:3,200, the rabbit was bled; if diaminobenzidine tetrahydrochloride (Sigma the titer was less, another booster was given to St. Louis, Mo.), 5 /xl 30% hydrogen peroxide the rabbit and the titer checked again after 2 wk. (Sigma), and 10 ml TBS were added to each disk. If the second titer remained low, the rabbit was After 30 min incubation at 37°C, the substrate discarded. solution was removed and each disk was air A portion of the serum was mixed with an dried. Disks were graded positive ( + ) or negaequal volume of saturated ammonium sulfate so- tive (—) in comparison with controls; those that lution. The precipitated globulin fraction was could not be confirmed positive or negative were dissolved in distilled water, and ammonium sul- graded as indeterminate (+/—). fate precipitation was repeated twice. The final solution of globulin was dialyzed extensively Results against 0.85% saline solution. The protein content of the solution was determined by a protein The results of the two techniques are preassay kit (Bio-Rad, Richmond, Calif.). sented in Table 1. Analyses by contingency taLabeling of Globulin with Fluorescein. The bles at the 95% significance level indicated that protein content of the globulin solution was ad- for noninfected chiggers, there was no relationjusted to 1% with normal saline. A volume of ship between test results and test technique (P > carbonate—bicarbonate buffer (0.5M, pH 9.0) 0.05); however, for infected chiggers, the relative equivalent to 10% of the volume of globulin so- proportions of test results between DFAT and lution was added. The buffered globulin solution DIBA (P < 0.05) were significantly different. was kept chilled, and 0.05 mg of fluorescein DIBA was found to be much more sensitive isothiocynate (Baltimore Biological Laborato- than DFAT (Table 2); however, their specificiries, Md.) per mg globulin was added slowly ties were similar. The negative predictive value with constant stirring. The solution was stirred of DIBA was better than that of DFAT. DFAT overnight, then dialyzed for 24 h against 0.85% had a higher false negative percentage. saline solution. Extensive dialysis against phosphate-buffered saline (0.01M phosphate, Discussion 0.15M NaCl, pH 7.5) was performed until excess fluorescein was removed completely. The conjuDIBA appears to be comparable to, if not betgate was cleared by centrifugation and stored at ter than, DFAT for the detection of scrub typhus -20°C. DFAT. The technique devised by Dohany et Table 2. Relative merits of direct fluorescent antibody al. (1978) was followed, and the fluoresceintechnique (DFAT) and dot-immunobinding assay (DIBA) labeled globulin prepared earlier was used. for detection of scrub typhus infection in L. fletcheri DIBA. Cellulose acetate disks (6 mm diameter) were soaked in distilled water for 5 min, then % Actual infectivity0 dried overnight. Each chigger was squashed in Parameter DFAT DIBA 2.5 ix\ of Tris-balanced salt solution (TBS) (0.05M + + Tris, 0.2M Nacl, pH 7.4). The exudate was trans73 5 92 6 ferred to a disk and dried overnight. Each disk + 20 91 5 91 then was soaked in 150 fi\ of blocking solution Sensitivity 78.5 94.8 (3% skimmed milk) for 1 h at 37°C. The blocking Specificity 94.8 93.8 solution was removed, and 150 /ul of rabbit hy- Positive predictive value 93.6 93.9 82.0 94.8 perimmune serum was added. Each disk was Negative predictive value positive percent 6.4 6.1 incubated at 37°C for 2 h. After removal of serum, False False negative percent 18.0 5.2 each disk was washed with TBS and 150 fi\ of goat anti-rabbit Ig (A + M + G) peroxidase con+ , positive; - , negative.

July 1992

HO ET AL: DOT-lMMUNOBINDING ASSAY FOR SCRUB TYPHUS

Acknowledgment The authors are grateful to the Director, Institute for Medical Research, Kuala Lumpur, for permission to publish this article. Assistance from the following personnel is greatly appreciated: T. C. Chan, P. T. Chang, Fadhilah Ishak, O. W. Phang, and Halimaton Ibrahim.

This study was funded by a research grant (3-08-01052) from the Malaysian Government.

References Cited Bozeman, F. M. & B. L. Elisberg. 1963. Serological diagnosis of scrub typhus by indirect immunofluorescence. Proc. Soc. Exp. Biol. Med. 112: 568-573. Dohany, A. L., A. Shirai, D. M. Robinson, S. Ram & D. L. Huxsoll. 1978. Identification and antigenic typing of Rickettsia tsutsugamushi in naturally infected chiggers (Acarina: Trombiculidae) by direct immunofluorescence. Am. J. Trop. Med. Hyg. 27: 1261-1264. Hawkes, R., E. Niday & J. Cordon. 1982. A dotimmunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119: 142-147. Oaks, Jr., S. C , R. L. Ridgway, A. Shirai & J. C. Twartz. 1983. Scrub typhus. Bull. Inst. Med. Res. Kuala Lumpur 21. Oprandy, J. J. & G. W. Long. 1990. Processing and microfiltration of mosquitoes for malaria antigen detection in a rapid dot immunobinding assay. J. Clin. Microbiol. 28: 1701-1703. Rapmund, G., R. W. Upham, W. D. Kundin, C. Manikumaran & T. C. Chan. 1969. Transovarial development of scrub typhus rickettsiae in a colony of vector mites. Trans. R. Soc. Trop. Med. Hyg. 63: 251-258. Robinson, D. M., G. Brown, E. Gan & D. L. Huxsoll. 1976. Adaptation of a microimmunofluorescence test to the study of human Rickettsia tsutsugamushi antibody. Am. J. Trop. Med. Hyg. 25: 900-905. Shirai, A., R. D. Montrey, R. M. Werner, S. Arimbalam & D. L. Huxsoll. 1979. Clinical responses of silvered leaf monkeys to infection with selected strains of Rickettsia tsutsugamushi. J. Infect. Dis. 140: 811-814. Shirai, A., D. L. Huxsoll, A. L. Dohany, R. D. Montrey, R. M. Werner & E. Gan. 1982. Characterisation of Rickettsia tsutsugamushi strains in two species of naturally infected laboratory-reared chiggers. Am. J. Trop. Med. Hyg. 31: 395-402. Tsai, S. J., L. J. Hutchinson & A. Zarkower. 1989. Comparison of dot immunobinding assay, enzymelinked immunosorbent assay and immunodiffusion for serodiagnosis of paratuberculosis. Can. J. Vet. Res. 53: 405-410. Woods, G. L. & P. C. Iwen. 1990. Comparison of a dot immunobinding assay, latex agglutination, and cytotoxin assay for laboratory diagnosis of Clostridium difficile-associated diarrhea. J. Clin. Microbiol. 28: 855-857. Received for publication 4 September 1991; accepted 13 January 1992.

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infections in L. fletcheri. The advantages of higher sensitivity, higher negative predictive value, and lower percentage false negative support the use of DIBA for routine screening of chiggers. Because it does not require any sophisticated equipment, the DIBA is most adaptable for use in a field test kit. Individual chiggers can be simultaneously infected with more than one strain of R. tsutsugamushi (Shirai et al. 1982). At the present stage of development, it is uncertain whether the DIBA is detecting only the Karp strain. Cross-reactions among hyperimmune sera to various strains of R. tsutsugamushi has been shown (Bozeman & Elisberg 1963). In the DFAT, by using dilutions of fluorescein conjugates in which such crossreactions are negated, strain typing of the rickettsia can be performed. The same can probably be done with the DIBA. Using fluorescein conjugates against nine strains of R. tsutsugamushi, Dohany et al. (1978) was able to achieve a sensitivity of 93.3% with the DFAT, in marked contrast to the sensitivity of 78.5% achieved in this study. It is suggested that the lower sensitivity was caused by the dilution of fluorescein conjugate used in this study; only those chiggers infected with Karp strain were detected. It had also been observed that infection with a particular strain may not be present in all chiggers of the same generation and line (T.M.H., unpublished data). Thus, it is possible that not all the chiggers of the infected population used in this study were infected with the Karp strain; such chiggers that were infected with other strains would give negative DFAT results in this study. In conclusion, it is apparent that the DIBA is a sensitive and specific technique for detecting scrub typhus infections in L. fletcheri. It is suitable for use under field conditions. Further investigations are required to make it suitable for strain typing and for use with other species of vector chiggers.

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Development and evaluation of dot-immunobinding assay for detection of scrub typhus infection in Leptotrombidium fletcheri (Acari: Trombiculidae).

A dot-immunobinding assay (DIBA) was compared with a direct fluorescent antibody technique (DFAT) for the detection of Rickettsia tsutsugamushi infect...
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