In Vitro Antibiotic Susceptibility of Orientia tsutsugamushi strain Boryong Measured by Flow Cytometry / 감염과화학요법

BACKGROUND: Scrub typhus, an infectious disease caused by Orientia tsutsugamushi, is endemic in Korea. With the introduction of tetracycline and chloramphenicol in clinical practice, the mortality due to scrub typhus has markedly decreased. In 1995, scrub typhus poorly responsive to doxycycline was reported in Thailand; the need for safe antibiotics for the treatment of scrub typhus acquired during pregnancy or for children is emerging; also, broader spectrum antibiotics having anti-Orientia activity may be preferred for empirical therapy of enteric fever syndrome and for complicated scrub typhus. The anti-Orientia activities of various antibiotics, including recently licensed antibiotics, were investigated by flow cytometry. MATERIALS AND METHODS: O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15, a monoclonal antibody reacting against a linear epitope on 56-kDa major outer membrane protein of O. tsutsugamushi. Then the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia). A concentration at which no further decrease in growth index occurred was defined as the minimal inhibitory concentration (MIC). Microbial susceptibilities to the following antibiotics were measured: quinupristin-dalfopristin (Synercid), levofloxacin, ciprofloxacin, moxifloxacin, metronidazole, linezolid, clindamycin, chloramphenicol, doxycycline, azithromycin, and rifampin. RESULTS: Considering the usual serum concentrations of rifampin (MIC=0.025-0.05 microg/mL), azithromycin (MIC=0.05-0.5 microg/mL) and doxycycline (MIC=0.05-0.1 microg/mL), these antibiotics exhibited very low MICs. Synercid (MIC=0.2-1.0 microg/mL), clindamycin (MIC=1.0 microg/mL) and chloramphenicol (MIC=1-2 microg/mL) exhibited moderately low MICs; moxifloxacin (MIC=8 microg/mL), ciprofloxacin (MIC=25.6 microg/mL or more) and levofloxacin (MIC=30 microg/mL) exhibited relatively high MICs; and cefotaxime (MIC>50 microg/mL), metronidazole (MIC>30 microg/mL) and linezolid (>30 microg/mL) exhibited high MICs. CONCLUSIONS: Among the new antibiotics, none was superior to doxycycline, azithromycin or rifampin with respect to anti-Orientia activity. Synercid, clindamycin, and moxifloxacin may show moderate therapeutic efficacies in human.

A Case of Scrub Typhus in Summer Presenting as Atypical Pneumonia / 감염과화학요법

Scrub typhus usually occurs in October and November in Korea. Its typical clinical manifestations are fever, rash, eschar, and lymphadenopathies. Pneumonitis may occur, however, it occurs as a complication at the late phase of scrub typhus, when it is inappropriately treated. We encountered a case of pneumonitis that was an initial manifestation of scrub typhus which was confirmed by rickettsial culture, positive seroconversion, and polymerase chain reaction. The case was a 16-year-old high school adolescent who presented with fever and headache that lasted for 1 day in July, 2007. He denied of any outdoor activities. A chest radiograph showed patchy infiltrates in the left lower lung, which improved with the administration of cefotaxime and azithromycin for empirical treatment of community-acquired pneumonia. Blood taken on the second febrile day showed a positive culture for Orientia tsutsugamushi and was also positive for O. tsutsugamushi DNA polymerase chain reaction. Immunofluorescent antibody (IFA) test for O. tsutsugamushi showed that the initial antibody was negative, but convalescent serum tested positive with an antibody titer of 1:80. IFA for Coxiella burnetii showed false positive results; the initial IgM and IgG titers were 1:128 and 1:256, respectively, and the IgM and IgG titers in convalescent serum were 1:128 and 1:128, respectively.

A Study of HHA-8 Seroprevalence in Korean Health Care Workers / 감염과화학요법

HHV-8 virus is known to be associated with Kaposi's sarcoma (KS) in HIV seropositive patients and its seroprevalence has geographic difference. In South Korea, incidence of KS is low not only in general population but also in HIV patients compared to other countries. It is speculated that low seroprevalence of HHV-8 has contributed to the low incidence of KS in South Korea. We examined the seroprevalence of anti HHV-8 IgG of HIV seronegative health care workers in one University hospital. Enzyme-linked immunosorbent assay(ELISA) was used for diagnosis. One person (0.6%, 1/164) was seropositive, six were equivocal (3.7%, 6/164) and 157 people were seronegative (95.73%, 157/164). This study revealed that seroprevalence of HHV-8 in general population was low in South Korea. Further studies are needed to be carried out to evaluate the low HHV-8 seroprevalence in Korea.

In vitro Study on the Dose and Duration of Doxycycline Treatment against Orientia tsutsugamushi / 감염과화학요법

BACKGROUND: Doxycycline, azithromycin, and chloramphenicol have been used for the treatment of scrub typhus; however, the duration of treatment for this disease is still controversial. Most clinical studies on the treatment of scrub typhus focus on the effectiveness of antibiotics; few studies have focused on the duration of the treatment. There has been no in vitro evaluation of the optimal duration of treatment of scrub typhus. We performed an experiment to evaluate the regrowth of Orientia tsutsugamushi after exposure to doxycycline of various concentration and exposure time. MATERIALS AND METHODS: O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15, a monoclonal antibody reacting against a linear epitope on the 56-kDa major outer membrane protein of O. tsutsugamushi; thereafter, the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia). Incubation in media containing 0.1 (the minimal inhibitory concentration of doxycycline against O. tsutsugamushi strain Boryong), 0.2, and 2 microg/mL doxycycline for 1, 3, 5, and 7 day was followed by incubation in antibiotic-free media for 5 day. Two inocula sizes were employed; low inoculum (45.18% of cells were infected) and high inoculum (87.21% of cells). RESULTS: The incubation with 0.1 microg/mL doxycycline resulted in O. tsutsugamushi regrowth irrespective of doxycycline treatment duration. When the concentration of doxycycline was increased to 0.2 or 2 microg/mL, regrowth was observed in short duration treatments (1-5 day at 0.2 microg/mL; 1 day at 2 microg/mL). Higher inoculum size of O. tsutsugamushi increased regrowth (1 day at the low inoculum; 1-5 day at the high inoculum). Conclusions: The regrowth of O. tsutsugamushi after doxycycline treatment depends on the doxycycline dose; the higher the dose, the lower the relapse rate. If this result is extrapolated to humans, higher dose of doxycycline might be preferred to lower the relapse rate. The inoculum effect in O. tsutsugamushi infection needs further investigations.

Doxycycline Resistance in Orientia tsutsugamushi Isolated from Korean Patients / 감염과화학요법

BACKGROUND: Doxycycline has been the drug of choice for the treatment of scrub typhus. However, scrub typhus with poor response to doxycycline was first reported in Thailand in 1996. To date, it is not known whether doxycycline resistant scrub typhus is present in Korea. To investigate the doxycycline resistance of scrub typhus in Korea, we performed an experiment to evaluate the degree of growth inhibition of Orientia tsutsugamushi at the minimal inhibitory concentration (MIC) of doxycycline. MATERIALS AND METHODS: 37 isolates of Orientia tsutsugamushi were collected from Korean patients with scrub typhus during 10 year period (from 1997 to 2007). 34 isolates consisted of Boryong serotype or related serotypes. These strains were stained with FS15, a monoclonal antibody against the linear epitope on the protein located at the outer membrane of O. tsutsugamushi. Three isolates were confirmed as serotype Gilliam, which was stained by anti-Gilliam antibody. The antibody-stainable 35 isolates were incubated in media containing 0.1 and 0.2 microg/mL of doxycycline for 3 days. Then the growth indices of Orientia were measured by flow cytometry. If the growth index was greater than 10%, the isolate was tentatively classified as an isolate resistant to doxycycline and its MIC was measured. RESULTS: Demographic and treatment data were similar to the results of previous reports. Infection was mainly observed in Inchon and the Chungcheongnam province. No patient showed delayed response to antibiotics. Of 35 hospitalized patients, 4 (11.4%) were managed at the intensive care unit, and 1 (2.9%) died of nosocomial pneumonia. All the 2 patients whose isolates were serotype Gilliam Yonchon/Ikeda-like strain were managed at the intensive care unit. Six of the 34 isolates from Korean patients exhibited growth indices greater than 10% at the MIC of doxycycline; however, the MICs of these isolates did not exceed 0.2 microg/mL. One isolate of serotype Gilliam showed an MIC of 0.0125 ug/mL, which seemed to be underestimated due to weak fluorescence of the anti-Gilliam antibody. Conclusions: Although the number of tested isolates is small, insensitivity of scrub typhus to doxycycline is not a major reason for treatment failure in Korea. Serotype Gilliam, especially Yochon/Ikeda-like strain, may be associated with severe form of scrub typhus. More studies concerning the antibiotic susceptibility for serotype Gilliam are warranted.

In vitro Efficacy of Antibiotic Combinations against Orientia tsutsugamushi / 감염과화학요법

BACKGROUND: Occasionally, combinations of antibiotics are used for the treatment of scrub typhus. However, the effectiveness of such combined therapies has rarely been evaluated. To date, no experimental studies have been performed; only 1 clinical study has assessed the efficacy of combined doxycycline and rifampin therapy. To elucidate the efficacies of other antibiotic combinations, we performed an experiment to evaluate the in vitro efficacy of antibiotic combinations against Orientia tsutsugamushi. MATERIALS AND METHODS: O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were cultured in antibiotic-containing media for 3-5 days and stained with FS15, a monoclonal antibody reacting against the linear epitope on the 56-kDa major outer membrane protein of O. tsutsugamushi. Thereafter, antimicrobial susceptibility was measured by flow cytometry and expressed as a growth index (total mass of Orientia). The growth indices of doxycycline (0.1 microg/mL), azithromycin (0.1 microg/mL), rifampin (0.0125 microg/mL), cefotaxime (2 and 20 microg/mL), and their various combinations (doxycycline+cefotaxime, doxycycline+rifampin, azithromycin+cefotaxime, and rifampin+cefotaxime) were measured. The above mentioned antibiotic concentrations, except for that of cefotaxime, represent the minimal inhibitory concentrations of each antibiotic. RESULTS: The growth indices of doxycycline (4.67% and 0.52%), rifampin (2.35% and 0.26%), and azithromycin (7.54%) were within the range of full suppression of O. tsutsugamushi; in contrast, cefotaxime (87.60%) was in effective. The growth indices of doxycycline+rifampin were 0.10% and 0.10%, which were similar to those obtained with doxycycline or rifampin alone. The growth indices of doxycycline+cefotaxime were 3.99% and 3.65% in low-dose cefotaxime (2 microg/mL), and 3.69% and 4.40% in high-dose cefotaxime (20 microg/mL). The growth indices of rifampin+cefotaxime (2.19% and 2.19% at 2 microg/mL; 1.84% and 2.04% at 20 microg/mL cefotaxime) were similar to those obtained with rifampin alone (2.35% and 0.26%). Azithromycin+cefotaxime (11.06-14.63%) showed higher growth indices than azithromycin alone; this suggests that this combination may be antagonistic. Conclusions: The anti-Orientia efficacies of doxycycline+rifampin, doxycycline+cefotaxime, and rifampin+cefotaxime were not antagonistic. The efficacy of the azithromycin+cefotaxime combination needs to be confirmed by more sensitive methods to exclude the possibility of antagonistic interactions between the antibiotics.

In Vitro Antibiotic Susceptibility of Orientia tsutsugamushi strain Boryong Measured by Flow Cytometry / 감염과화학요법

BACKGROUND: Scrub typhus, an infectious disease caused by Orientia tsutsugamushi, is endemic in Korea. With the introduction of tetracycline and chloramphenicol in clinical practice, the mortality due to scrub typhus has markedly decreased. In 1995, scrub typhus poorly responsive to doxycycline was reported in Thailand; the need for safe antibiotics for the treatment of scrub typhus acquired during pregnancy or for children is emerging; also, broader spectrum antibiotics having anti-Orientia activity may be preferred for empirical therapy of enteric fever syndrome and for complicated scrub typhus. The anti-Orientia activities of various antibiotics, including recently licensed antibiotics, were investigated by flow cytometry. MATERIALS AND METHODS: O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15, a monoclonal antibody reacting against a linear epitope on 56-kDa major outer membrane protein of O. tsutsugamushi. Then the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia). A concentration at which no further decrease in growth index occurred was defined as the minimal inhibitory concentration (MIC). Microbial susceptibilities to the following antibiotics were measured: quinupristin-dalfopristin (Synercid), levofloxacin, ciprofloxacin, moxifloxacin, metronidazole, linezolid, clindamycin, chloramphenicol, doxycycline, azithromycin, and rifampin. RESULTS: Considering the usual serum concentrations of rifampin (MIC=0.025-0.05 microg/mL), azithromycin (MIC=0.05-0.5 microg/mL) and doxycycline (MIC=0.05-0.1 microg/mL), these antibiotics exhibited very low MICs. Synercid (MIC=0.2-1.0 microg/mL), clindamycin (MIC=1.0 microg/mL) and chloramphenicol (MIC=1-2 microg/mL) exhibited moderately low MICs; moxifloxacin (MIC=8 microg/mL), ciprofloxacin (MIC=25.6 microg/mL or more) and levofloxacin (MIC=30 microg/mL) exhibited relatively high MICs; and cefotaxime (MIC>50 microg/mL), metronidazole (MIC>30 microg/mL) and linezolid (>30 microg/mL) exhibited high MICs. CONCLUSIONS: Among the new antibiotics, none was superior to doxycycline, azithromycin or rifampin with respect to anti-Orientia activity. Synercid, clindamycin, and moxifloxacin may show moderate therapeutic efficacies in human.

A Case of Scrub Typhus in Summer Presenting as Atypical Pneumonia / 감염과화학요법

Scrub typhus usually occurs in October and November in Korea. Its typical clinical manifestations are fever, rash, eschar, and lymphadenopathies. Pneumonitis may occur, however, it occurs as a complication at the late phase of scrub typhus, when it is inappropriately treated. We encountered a case of pneumonitis that was an initial manifestation of scrub typhus which was confirmed by rickettsial culture, positive seroconversion, and polymerase chain reaction. The case was a 16-year-old high school adolescent who presented with fever and headache that lasted for 1 day in July, 2007. He denied of any outdoor activities. A chest radiograph showed patchy infiltrates in the left lower lung, which improved with the administration of cefotaxime and azithromycin for empirical treatment of community-acquired pneumonia. Blood taken on the second febrile day showed a positive culture for Orientia tsutsugamushi and was also positive for O. tsutsugamushi DNA polymerase chain reaction. Immunofluorescent antibody (IFA) test for O. tsutsugamushi showed that the initial antibody was negative, but convalescent serum tested positive with an antibody titer of 1:80. IFA for Coxiella burnetii showed false positive results; the initial IgM and IgG titers were 1:128 and 1:256, respectively, and the IgM and IgG titers in convalescent serum were 1:128 and 1:128, respectively.

A Study of HHA-8 Seroprevalence in Korean Health Care Workers / 감염과화학요법

HHV-8 virus is known to be associated with Kaposi's sarcoma (KS) in HIV seropositive patients and its seroprevalence has geographic difference. In South Korea, incidence of KS is low not only in general population but also in HIV patients compared to other countries. It is speculated that low seroprevalence of HHV-8 has contributed to the low incidence of KS in South Korea. We examined the seroprevalence of anti HHV-8 IgG of HIV seronegative health care workers in one University hospital. Enzyme-linked immunosorbent assay(ELISA) was used for diagnosis. One person (0.6%, 1/164) was seropositive, six were equivocal (3.7%, 6/164) and 157 people were seronegative (95.73%, 157/164). This study revealed that seroprevalence of HHV-8 in general population was low in South Korea. Further studies are needed to be carried out to evaluate the low HHV-8 seroprevalence in Korea.

In vitro Study on the Dose and Duration of Doxycycline Treatment against Orientia tsutsugamushi / 감염과화학요법

BACKGROUND: Doxycycline, azithromycin, and chloramphenicol have been used for the treatment of scrub typhus; however, the duration of treatment for this disease is still controversial. Most clinical studies on the treatment of scrub typhus focus on the effectiveness of antibiotics; few studies have focused on the duration of the treatment. There has been no in vitro evaluation of the optimal duration of treatment of scrub typhus. We performed an experiment to evaluate the regrowth of Orientia tsutsugamushi after exposure to doxycycline of various concentration and exposure time. MATERIALS AND METHODS: O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15, a monoclonal antibody reacting against a linear epitope on the 56-kDa major outer membrane protein of O. tsutsugamushi; thereafter, the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia). Incubation in media containing 0.1 (the minimal inhibitory concentration of doxycycline against O. tsutsugamushi strain Boryong), 0.2, and 2 microg/mL doxycycline for 1, 3, 5, and 7 day was followed by incubation in antibiotic-free media for 5 day. Two inocula sizes were employed; low inoculum (45.18% of cells were infected) and high inoculum (87.21% of cells). RESULTS: The incubation with 0.1 microg/mL doxycycline resulted in O. tsutsugamushi regrowth irrespective of doxycycline treatment duration. When the concentration of doxycycline was increased to 0.2 or 2 microg/mL, regrowth was observed in short duration treatments (1-5 day at 0.2 microg/mL; 1 day at 2 microg/mL). Higher inoculum size of O. tsutsugamushi increased regrowth (1 day at the low inoculum; 1-5 day at the high inoculum). Conclusions: The regrowth of O. tsutsugamushi after doxycycline treatment depends on the doxycycline dose; the higher the dose, the lower the relapse rate. If this result is extrapolated to humans, higher dose of doxycycline might be preferred to lower the relapse rate. The inoculum effect in O. tsutsugamushi infection needs further investigations.

Doxycycline Resistance in Orientia tsutsugamushi Isolated from Korean Patients / 감염과화학요법

BACKGROUND: Doxycycline has been the drug of choice for the treatment of scrub typhus. However, scrub typhus with poor response to doxycycline was first reported in Thailand in 1996. To date, it is not known whether doxycycline resistant scrub typhus is present in Korea. To investigate the doxycycline resistance of scrub typhus in Korea, we performed an experiment to evaluate the degree of growth inhibition of Orientia tsutsugamushi at the minimal inhibitory concentration (MIC) of doxycycline. MATERIALS AND METHODS: 37 isolates of Orientia tsutsugamushi were collected from Korean patients with scrub typhus during 10 year period (from 1997 to 2007). 34 isolates consisted of Boryong serotype or related serotypes. These strains were stained with FS15, a monoclonal antibody against the linear epitope on the protein located at the outer membrane of O. tsutsugamushi. Three isolates were confirmed as serotype Gilliam, which was stained by anti-Gilliam antibody. The antibody-stainable 35 isolates were incubated in media containing 0.1 and 0.2 microg/mL of doxycycline for 3 days. Then the growth indices of Orientia were measured by flow cytometry. If the growth index was greater than 10%, the isolate was tentatively classified as an isolate resistant to doxycycline and its MIC was measured. RESULTS: Demographic and treatment data were similar to the results of previous reports. Infection was mainly observed in Inchon and the Chungcheongnam province. No patient showed delayed response to antibiotics. Of 35 hospitalized patients, 4 (11.4%) were managed at the intensive care unit, and 1 (2.9%) died of nosocomial pneumonia. All the 2 patients whose isolates were serotype Gilliam Yonchon/Ikeda-like strain were managed at the intensive care unit. Six of the 34 isolates from Korean patients exhibited growth indices greater than 10% at the MIC of doxycycline; however, the MICs of these isolates did not exceed 0.2 microg/mL. One isolate of serotype Gilliam showed an MIC of 0.0125 ug/mL, which seemed to be underestimated due to weak fluorescence of the anti-Gilliam antibody. Conclusions: Although the number of tested isolates is small, insensitivity of scrub typhus to doxycycline is not a major reason for treatment failure in Korea. Serotype Gilliam, especially Yochon/Ikeda-like strain, may be associated with severe form of scrub typhus. More studies concerning the antibiotic susceptibility for serotype Gilliam are warranted.

In vitro Efficacy of Antibiotic Combinations against Orientia tsutsugamushi / 감염과화학요법

BACKGROUND: Occasionally, combinations of antibiotics are used for the treatment of scrub typhus. However, the effectiveness of such combined therapies has rarely been evaluated. To date, no experimental studies have been performed; only 1 clinical study has assessed the efficacy of combined doxycycline and rifampin therapy. To elucidate the efficacies of other antibiotic combinations, we performed an experiment to evaluate the in vitro efficacy of antibiotic combinations against Orientia tsutsugamushi. MATERIALS AND METHODS: O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were cultured in antibiotic-containing media for 3-5 days and stained with FS15, a monoclonal antibody reacting against the linear epitope on the 56-kDa major outer membrane protein of O. tsutsugamushi. Thereafter, antimicrobial susceptibility was measured by flow cytometry and expressed as a growth index (total mass of Orientia). The growth indices of doxycycline (0.1 microg/mL), azithromycin (0.1 microg/mL), rifampin (0.0125 microg/mL), cefotaxime (2 and 20 microg/mL), and their various combinations (doxycycline+cefotaxime, doxycycline+rifampin, azithromycin+cefotaxime, and rifampin+cefotaxime) were measured. The above mentioned antibiotic concentrations, except for that of cefotaxime, represent the minimal inhibitory concentrations of each antibiotic. RESULTS: The growth indices of doxycycline (4.67% and 0.52%), rifampin (2.35% and 0.26%), and azithromycin (7.54%) were within the range of full suppression of O. tsutsugamushi; in contrast, cefotaxime (87.60%) was in effective. The growth indices of doxycycline+rifampin were 0.10% and 0.10%, which were similar to those obtained with doxycycline or rifampin alone. The growth indices of doxycycline+cefotaxime were 3.99% and 3.65% in low-dose cefotaxime (2 microg/mL), and 3.69% and 4.40% in high-dose cefotaxime (20 microg/mL). The growth indices of rifampin+cefotaxime (2.19% and 2.19% at 2 microg/mL; 1.84% and 2.04% at 20 microg/mL cefotaxime) were similar to those obtained with rifampin alone (2.35% and 0.26%). Azithromycin+cefotaxime (11.06-14.63%) showed higher growth indices than azithromycin alone; this suggests that this combination may be antagonistic. Conclusions: The anti-Orientia efficacies of doxycycline+rifampin, doxycycline+cefotaxime, and rifampin+cefotaxime were not antagonistic. The efficacy of the azithromycin+cefotaxime combination needs to be confirmed by more sensitive methods to exclude the possibility of antagonistic interactions between the antibiotics.