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Journal of Clinical Microbiology, August 2005, p. 4306-4307, Vol. 43, No. 8
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.8.4306-4307.2005
| LETTER TO THE EDITOR |
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LETTER |
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This study was focused on Korean patients with acute febrile illness from December 1992 to November 1993. The diagnosis was made on the basis of indirect immunofluorescence assay in which only Rickettsia japonica and Orientia tsutsugamushi were used as rickettsial antigens. Overall, 676 (19.88%) out of 3,401 patients were found to have polyvalent antibody at a 1:40 serum dilution against R. japonica. In another study by the same group with the same study design, 546 (16.24%), 482 (14.34%), and 269 (8%) out of 3,362 patients were seropositive at the level of 1:40 dilution against R. akari, R. conorii, and R. sibirica, respectively (2). Because these serum samples of both studies were obtained from the same population from 1992 to 1993, there is significant overlapping in populations who were seropositive to individual SFG rickettsial antigens, obviously. However, the authors did not consider the effect of cross-reactions between R. japonica and other SFG and typhus group rickettsiae. They did not analyze the antibody titers of other rickettsial antigens except O. tsutsugamushi in this study.
In a recent study, the authors further demonstrated the existence of several SFG rickettsiae in sera of Korean patients by using a PCR-based amplification method (3). They collected 200 serum samples with immunoglobulin M titers of 1:40 to 1:160 against SFG rickettsial antigens from 1993 to 1999. Only one among them showed a sequence homologous to that of the partial rickettsial outer membrane protein B gene (rompB) of R. japonica. The significant discrepancy between results of serology and molecular methods may be caused by inadequate cutoff values and cross-reactions. It is reasonable that a significant part of these 676 seropositive patients should not be ascribed to JSF and further confirmatory tests are necessary. In other words, they have overestimated the seroprevalence of JSF in Korea and the epidemiological analysis which they made accordingly is inadequate. Though Paddock et al. have mentioned the concept, we would like to emphasize again that the serological tests are only group-specific and cannot be used to ascribe etiology to a specific pathogen of SFG rickettsiae (4).
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Present address: 615 Calibre Woods Drive N.E., Atlanta, GA 30329. 
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Jui-Shan Ma ,*
Department of Pediatrics Show-Chwan Memorial Hospital 542, Sec. 1 Chung-Shan Rd. Changhua 500, Taiwan ext. 2007 Fax: (886) 4-723-6226 E-mail: morrison@show.org.tw
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In our study, we conducted several serosurveys of SFG and TG rickettsiae in Korean patients with acute febrile illness from December 1992 to November 1993 by immunofluorescence assay (IFA) (3, 4). Dr. Ma correctly writes that there was significant overlapping among populations that were seropositive to individual SFG rickettsial antigens. We showed serological cross-reactions between the three SFG rickettsiae (R. akari, R. conorii, and R. sibirica) and two TG rickettsiae (R. typhi and R. prowazekii) (4). However, we did not show the cross-reactions between R. japonica and other SFG and TG rickettsiae in our report (3). Therefore, we would like to present the data of the cross-reactions between the above-mentioned rickettsiae in the overlapped serum samples. Of the 3,362 sera tested by IFA, the number of polyvalent positive sera against R. japonica at a 1:40 serum dilution was 662 (19.69%). Among these 662 sera, 380 were also seropositive against any of the other SFG rickettsiae (R. akari, R. conorii, and R. sibirica) and 307 were also seropositive to either one of the two TG rickettsiae (R. typhi and R. prowazekii). The number of sera that had no antibodies against any other SFG and TG rickettsiae was 195 (5.80%) out of the 3,362 sera.
Recently, using a PCR-based amplification method, we demonstrated the existence of nucleic acid sequences of SFG rickettsiae in serum specimens that were seropositive for SFG rickettsiae (1). Sequence analysis of the PCR products showed that 23 sera contained sequences that were homologous to the sequence of SFG rickettsiae. Among them, 17 sequences were homologous to that of R. conorii, 2 were homologous to that of R. akari, 1 was homologous to that of R. japonica, and 3 were homologous to that of R. felis (1). In his letter, Dr. Ma stresses that only one of the sequences was homologous to that of R. japonica and suggests that the significant discrepancy between the serological results and the molecular methods may be caused by inadequate cutoff values and cross-reactions. Dr. Ma also suggests that a significant part of these 676 seropositive patients should not be ascribed to Japanese spotted fever and that further confirmatory tests are necessary. We selected a screening IFA cutoff value of 
1:40 because the serum samples tested in our study were obtained from patients experiencing the early stage of febrile illness. The IFA results demonstrated that 676 out of the 3,401 tested sera contained polyvalent antibodies against R. japonica (3). As expected, many of the positive sera contained immunoglobulin M (IgM) antibodies (n = 377) rather than IgG antibodies (n = 167). For the diagnosis of rickettsial diseases, an IFA was considered positive if the R. coronii-specific IgG titers were 128 and/or IgM titers were 64, and in the case of other specific antigens, if the IgG titers were 64 and/or IgM titers were 32 (6). Even applying the criteria, 398 samples were found seropositive for R. japonica (11.70%). Based on these results, cross-reactions between R. japonica and other SFG and TG rickettsiae and the selection of a low cutoff value may explain the result of high seroprevalence in our serologic study, as Dr. Ma suggested. However, we think that the discrepancy between the serological results and molecular methods is due to the selection of primers for the PCR study rather than the inadequate cutoff values and cross-reactions. In the PCR study, we focused on not only detection but also identification of the minute rickettsial agents in serum samples. Therefore, we selected the primers derived from rather variable regions based on a multiple-sequence alignment of rickettsial outer membrane protein B gene (rompB) sequences. The primers showed the limitation of detection of the rickettsial agents in clinical samples. Recently we did further confirmatory tests to solve this. We designed specific primers that targeted conserved regions of rompB genes for the development of diagnostic tool for SFG rickettisosis. In our study, DNA templates from 71 out of 100 seropositive sera contained IgM antibodies against SFG rickettsiae and 19 out of 100 seronegative sera among acute febrile disease patients were amplified by nested PCR assay with the primers. Of the 90 positive amplicons, 25 (27.78%) were proved to be partial rompB genes of R. japonica by restriction fragment length polymorphism and sequencing analysis. The others were those of R. conorii, R. akari, and R. sibirica (2).
Even though, as suggested by Dr. Ma, serological assays have several limitations, it cannot be denied that the IFA method is the recognized standard for the diagnosis of rickettsioses and is used as a reference technique in most laboratories (5). We also believe that IFA is a reasonable approach to screening many clinical samples, such as the 3,401 sera used for our seroepidemiologic study of SFG rickettsioses. In conclusion, we still believe that our study showed the existence of SFG rickettsioses, especially Japanese spotted fever in Korea, and that the results of our succeeding studies adequately supported this claim.
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Won-Jong Jang Yeon-Joo Choi Kyung-Hee Park* Institute of Biomedical Science and Technology Department of Microbiology School of Medicine Konkuk University Choongju-si, Choongbuk, 380-701 Korea Fax: (82) 43-851-9329 E-mail: Kyunghee.park{at}kku.ac.kr
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