Improvement of the Diagnostic Sensitivity of Scrub Typhus Using a Mixture of Recombinant Antigens Derived from Orientia tsutsugamushi Serotypes

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.

Characterization of Korean Leptospira interrogans Isolates by Pulsed-Field Gel Electrophoresis of Not I Digests of DNA

BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.

Characterization of Korean Leptospira interrogans Isolates by Pulsed-Field Gel Electrophoresis of Not I Digests of DNA

BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.

Detection of Leptospiral DNA from Field Rodents by PCR

Leptospirosis has been one of important epidemic diseases in Korea since 1984. Wild rodents, mostly Apodemus agrarius, served the important source of infection especially in harvest season in rural areas of Korea. Prevalence of Leptospira spp. infection in field rodents were investigated by detecting leptospiral DNA from rodent kidney. Among 108 rodents collected from various areas in 1998, leptospiral DNA were detected from 7 rodents (6.48%). Among 104 rodents, Apodemus agrarius, captured from Yeonchon and Paju area in 2001 and 2002, leptospiral DNA were detected from 6 rodents (5.76%). No leptospiral DNA was detected from 23 rodonts (Apodemus peninsulae 16, Apodemus agrarius 2 and Eothenomys regulus 5) captured in Odae mountain area in 1998.

Characterization of a Species-specific Antigen in Rickettsia typhi

Murine typhus is an acute febrile illness caused by Rickettsia typhi. It is one of the four major acute febrile illnesses in Korea during autumn. To study a species-specific antigen of R. typhi, two clinical isolates (87-91 and 87-100) and two reference strains (VR-144 and VR-738) were analyzed by mouse antisera and monoclonal antibodies (MAbs). On SDS- polyacrylamide gel electrophoresis (PAGE), R. typhi showed major antigen bands of 135, 80, 75, 64, 47, 22, and 19 kDa and these bands differed with those of other species. On Western blot analysis, the MAbs reacting only with R. typhi could only detect 135 kDa protein. The 135 kDa protein appeared to be the species-specific antigen. Other MAbs showing cross-reactivity with R. prowazekii reacted with 135 kDa protein in fresh culture supernatant of R. typhi infected host cell. However, the cross-reacting antibody did also react with smaller protein bands, most of which seem to be degradation products of the 135 kDa protein since they increase in old protein stocks purified from R. typhi harvested from infected host cell. These suggest that 135 kDa protein is unstable and the R. typhi specific epitopes are located at the regions of 135 kDa protein that are removed when the protein is degraded. The 135 kDa protein or its specific and stable recombinant protein would serve an important target for the development of vaccine and specific diagnostic antigen.