INTRODUCTION
Scrub typhus is a febrile disease, endemic to the Asia-Pacific region, with case fatality rates up to 50% in untreated patients.1 It is a common cause of disease among indigenous peoples where up to 19.3% of all illnesses2 and up to 23% of febrile illnesses3 among hospitalized patients are due to scrub typhus. The disease is caused by infection with Orientia tsutsugamushi (formerly Rickettsia tsutsugamushi), which is transmitted to humans by the bite of an infected Leptotrombidium mite (chigger stage). Signs and symptoms are consistent with a systemic infection in which, fever, severe headache, and rash are commonly reported. Other signs and symptoms such as lymphadenopathy, myalgias, cough, sore throat, abdominal pain, stupor, and central nervous system involvement may also be seen.4,5 Scrub typhus is not only an important health risk factor for individuals living in the endemic region, but it is especially problematic for military personnel operating in endemic areas.4,6–8
Since 1934, outbreaks of scrub typhus have occurred sporadically among military personnel training at Camp Fuji, near Mount Fuji, Japan.8 In 1948, approximately 1.5% of 1,769 U.S. Army troops were admitted to Army hospitals with scrub typhus. In 1953, 57 cases of scrub typhus were diagnosed in two regiments of U.S. Marines. Throughout the period 1981–1983, 56 additional cases occurred among U.S. Marines. Six cases in more than 800 marines and sailors occurred within a three-week period during training exercises from October to November 1995.9 Japanese military forces also reported scrub cases among its personnel in 1934, 1959, and 1968.
Most recently, scrub typhus outbreaks have occurred in 2000 and 2001 with ten and nine individuals sick with suspected scrub typhus among approximately 800 and 900 U.S. Marines training at Camp Fuji, respectively.10,11 The area’s primary season for scrub typhus is September through December, which corresponds with its rainy season.8,10,11 There have been no cases reported of scrub typhus during this time frame for 2002 (Carte JJ, U.S. Marine Corps, Camp Fuji, Japan, unpublished data).
In this report, we describe the laboratory analysis of sera from 18 suspected cases and 46 controls from the two most recent outbreaks of scrub typhus at Camp Fuji using our standard trivalent (Karp, Kato, Gilliam) whole cell enzyme-linked immunosorbent assay (KpKtGm-wc ELISA),12 and evaluating new assays using recombinant proteins: a cassette format rapid lateral flow assay (RCT) and an ELISA (Kp r56 ELISA) both using Kp recombinant 56-kD protein (r56),13–15 and an ELISA and Western blot assay using r56 from the Karp, Kato, and Gilliam strains of O. tsutsugamushi (KpKtGm r56 ELISA and KpKtGm r56 Western blot, respectively). Recent studies with r56 from Karp in a passive hemagglutination assay,16 ELISA,13,17,18 and RCTs14,15,18 have shown the utility of this new reagent when compared with previous assays that use whole cell antigens, including an ELISA, an indirect immunofluorescent antibody test, an indirect immunoperoxidase test , and a dot-blot immunoassay. The addition of r56 from other strains of O. tsutsugamushi, such as Gilliam and Boryong, has also proven effective.16 In this investigation, r56 from the Gilliam and Kato strains have increased the sensitivity/specificity of our new ELISA and Western blot assays over that of the monovalent Kp r56 rapid flow assay (RFA) and the Kp r56 ELISA, and our previous laboratory standard (KpKtGm-wc ELISA).
MATERIALS AND METHODS
Outbreak site and personnel.
Camp Fuji is a military training area near Mount Fuji, Japan used extensively by the U.S. Marine Corps for field training. Approximately 800 U.S. Marines from the 3rd Battalion/3rd Marine Infantry Division, Marine Corps Base Hawaii trained at Camp Fuji from October 1 to November 10, 2000. The following year, from October 11 through November 9, 2001, approximately 900 members of the 1st Battalion/3rd Marine Regiment from Marine Corps Base Hawaii trained at Camp Fuji in the same general location. The exact locations where the mite contact occurred were impossible to ascertain for either group because the Marines moved constantly throughout the day, and slept in individual bedrolls on the ground in different locations every night.
Case definition.
The case definition for the 2000 outbreak was a Marine who trained at Camp Fuji between October 1 and November 10, 2000 and developed an acute illness between October 7 and December 1, 2000 that included at least two of the following signs or symptoms: fever (objective or subjective), rash, or eschar.
The case definition for the 2001 outbreak was a Marine who trained at Camp Fuji between October 11 and November 9, 2001 and developed an acute illness between October 17 and November 30, 2001 that included at least three of the following signs or symptoms: fever (objective or subjective), headache, rash, eschar, localized lymphadenopathy, or myalgia.
All suspected cases received doxycycline treatment to control the cause of their febrile illness.
Laboratory confirmation.
Acute sera from nine cases during the 2000 outbreak were tested at a Japanese civilian laboratory (Biomedical Laboratory, Tokyo, Japan) and were reportedly negative for scrub typhus; however, further review of the results indicated mildly positive titers in two patients (Table 1). Subsequently, convalescent sera were obtained between November 29 and December 6, 2000 and sent to the Naval Medical Research Center (NMRC) in Silver Spring, Maryland for testing. No acute sera were obtained for the 2001 outbreak. Convalescent sera were obtained in late January 2002 (except for one which was obtained on March 6, 2002) and sent to the NMRC. Confirmation of scrub typhus was a convalescent titer for O. tsutsugamushi ≥ 100 and a total absorbance value ≥ 1.00.
Serology.
ELISA.
The ELISA whole cell antigen (a whole cell preparation of 0.1 μg/well each of the Karp, Kato, and Gilliam strains of O. tsutsugamushi) and r56 (0.1 μg/well each of the Karp, Kato, and Gilliam strains) were allowed to coat microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) for two days at 4°C. Serum samples diluted four-fold from 1:100 to 1:6,400 and tested in triplicate were incubated at room temperature in microtiter wells with and without antigen. Antibody (IgG) specific to scrub typhus antigens were bound to immobilized antigen and detected by addition of goat anti-human IgG conjugate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). After a peroxidase substrate solution (2,2’-azino-di-[3-ethylbenzthiazoline sulfonate] [ABTS®]; Kirkegaard & Perry Laboratories) was added, a blue-green product was produced in wells where antibodies had been bound to the antigens. Concentrations of antibodies were measured at 405 nm by a Vmax/Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA). A positive and three negative control sera were run with each experiment.
A positive result was defined as detecting a net absorbance of the serum sample greater than the mean of three negative control sera plus three times their standard deviation or an absorbance of 0.200 (whichever was greater). The titer was determined to be the inverse of the highest serum dilution that produced a net absorbance of 0.200 or greater. A negative result was defined as a net absorbance of less than the mean of three negative control sera plus three times the standard deviation.
Scrub typhus RCT.
The RCT consists of a single strip with IgG and IgM tests on the same side of the strip in the cassette. The assay was performed according to the manufacturer’s instructions (PANBIO Limited, Brisbane, Australia). The Kp r56 protein was conjugated to gold particles as the indicator system. Five microliters of serum was added to the cassette strip, followed by two drops of buffer, and the result was read after 10 minutes.
A positive result was a purple color on the control line and on the test lines of the cassette strip for either IgG or IgM. A negative result was a purple color only on control line of the cassette strip. The control line was purple for all tests conducted and was required for validity of the assays according to the manufacturer’s instructions.15
Western blot.
The ELISA- and RFA-positive serum samples were assessed by Western blot analysis. The assay used 1 μg/gel of r56 from each of the Karp, Kato and Gilliam strains. These recombinant proteins were applied to a 10% sodium dodecyl sulfate-polyacrylamide one-lane mini-gel. After electrophoresis, the antigen was transferred from the gel to a nitrocellulose membrane (Millipore Corporation, Bedford, MA). The membrane blot was blocked from non-specific binding by incubation with a 10% skim milk blocking buffer (10% skim milk; Difco Laboratories Inc, Detroit, MI and phosphate-buffered saline; Sigma, St. Louis, MO) overnight at 4°C with shaking. The membrane was then incubated with sera diluted 1:100 at room temperature with shaking for one hour, followed by a wash step. The IgG bound to r56 was detected by addition of goat anti-human IgG conjugate (Kirkegaard and Perry Laboratories) at a dilution of 1:50,000. Following incubation for 30 minutes at room temperature and a wash step, the blot was developed with a chemiluminescent substrate solution (Pierce, Rockford, IL). A positive and a negative control serum sample were run in each experiment.
A positive result was the presence of a 41-kD band (for the truncated 56-kD recombinant protein) on the film. A negative result was the absence of the 41-kD band.
RESULTS
The 2000 outbreak.
Ten suspected scrub typhus cases with onset of symptoms between October 25 and November 3, 2000 reported to the Battalion Aid Station at Camp Fuji, Japan. All cases had fever, rash, and various other symptoms consistent with scrub typhus; eight had a definite eschar (Table 1). Convalescent serum samples were collected from nine suspected cases and 17 controls (individuals training at the same time and place as the cases, but without symptoms) approximately one month after the outbreak occurred and tested for antibodies to O. tsutsugamushi. These results are shown in Table 2 and Figure 1. Sera from six suspected cases were reactive to whole cell antigen preparations and nine were reactive to KpKtGm r56 antigen preparations from the O. tsutsugamushi Karp, Kato, and Gilliam strains by IgG ELISA. All 17 control sera were non-reactive in these tests. Four sera were reactive to Kp r56 ELISA antigen preparations and nine were positive by RCT. All 17 control sera were non-reactive. Nine sera were IgM reactive in the RCT. All control sera were again non-reactive. Western blot analysis with Kp r56, Kt r56, and Gm r56 antigens confirmed the presence of antibody to O. tsutsugamushi in all but one of the suspected cases. Serum from case 6 was negative in all assays, except for the RCT and KpKtGm r56 ELISA, and had a one plus positive response on the Gm r56 Western blot. It is possible that this individual had scrub typhus, but had a diminished antibody response due to the early administration of doxycycline (two days after onset of symptoms).
The 2001 outbreak.
Convalescent sera were collected from nine suspect cases (eight marines met the case definition; one marine, case 2, did not) and 29 controls (individuals training at the same time and place as the cases, but without symptoms) approximately three months after the outbreak occurred and tested for reactivity to various O. tsutsugamushi antigens. The results of the serologic analysis are shown in Table 3 and Figure 1. Sera from five cases were reactive to the KpKtGm-wc ELISA antigens and eight were reactive to the KpKtGM r56 ELISA antigens. All 29 control sera were non-reactive to these two antigen preparations. Four case sera were reactive to the single Kp r56 antigen in the ELISA and six were reactive in the RCT. All control sera were non-reactive to these tests. Western blot analyses (Kp r56, Kt r56, Gm r56) confirmed the reactivity of the case sera to O. tsutsugamushi antigens. Samples with higher ELISA titers had stronger staining bands on the Western blots (Table 3). The serum from case 2 was non-reactive in all scrub typhus serological assays performed. Case 2 was the only suspected case that did not meet the symptoms/signs criteria. Cases 1 and 4 both had low seroreactivity, which was observed only in the KpKtGm r56 ELISA and Western blot assays. The reason for this low reactivity is unclear. Both individuals met the signs/ symptoms criteria, and in fact, their signs/symptoms included both rash and eschar, which are highly suggestive for scrub typhus. Both subjects received doxycycline at least six days after symptom onset, so a diminished antibody response due to early administration of antibiotic does not appear to be a plausible explanation.
DISCUSSION
Scrub typhus is a common disease in Asia in both indigenous2,3,19–21 and visiting individuals.4,6–9,21–23 The disease is difficult to diagnose because the signs and symptoms are non-specific and the availability of specific laboratory assays is inconsistent. The lack of diagnostic assay accessibility is due to the need for reagents derived from O. tsutsugamushi, an obligate intracellular pathogen that is difficult to grow, the cost of producing the reagents, and the requirement for bio-safety level (BSL) 3 facilities to produce whole cell antigens. For these reasons, many laboratories throughout the endemic region do not develop their own assays or will not pay the high cost for commercial assays. Therefore, clinicians may not have access to laboratories that use scrub typhus-specific assays or the laboratories may use the nonspecific and insensitive Weil-Felix reaction that uses cross-reactive Proteus antigens to diagnose rickettsial diseases.
Fortunately, in the last 10 years several laboratories have developed scrub typhus assays that use recombinant proteins derived from O. tsutsugamushi. These antigens are produced by Escherichia coli in a BSL 2 laboratory, and have been found to be very consistent from lot to lot and are quite stable.14 The antigen of choice has been the immunodominant outer membrane 56-kD protein from various strains of O. tsutsugamushi (Karp, Kato, Gilliam, Boryong) either used singularly or in combination. In nature, the protein is produced in large amounts (approximately 15% of the cell’s protein) by O. tsutsugamushi and is strongly recognized by the immune systems of various hosts, including humans.24 The r56 antigen has been used successfully as a fused protein in passive hemagglutination assays16 and an ELISA,17 and in a truncated format in an ELISA and RFA.13–15
In this report, we evaluated the ability of a new assay (KpKtGm r56 ELISA) to diagnose scrub typhus during an outbreak investigation utilizing sera collected from U.S. Marines training in Japan during two outbreaks of scrub typhus.10,11 The recombinant protein antigens used in this assay were developed from the Karp, Kato, and Gilliam strains of O. tsutsugamushi. These strains were initially isolated from geographically divergent areas (New Guinea, Japan, and Burma, respectively), and are antigenically distinct from one another. This is the first time that these non-fusion, truncated, recombinant proteins have been used together in a diagnostic assay. The assay (KpKtGm r56 ELISA) was evaluated against our standard ELISA that uses whole cell antigens from the Karp, Kato and Gilliam strains (KpKtGm-wc ELISA)12 and our recently developed ELISA using the monovalent Karp r56 antigen.13 In addition, we compared the KpKtGm r56 ELISA to an improved version of a cassette format lateral flow assay from PANBIO.15
The results of this study showed that all 17 case sera collected from individuals involved in two recent scrub typhus outbreaks among U.S. Marines training at Camp Fuji, Japan in 2000 and 2001 were positive when tested with the new trivalent KpKtGm r56 ELISA. In contrast, only 11 of the same sera set were positive by the KpKtGm-wc ELISA, eight were positive by the Kp r56 ELISA, and 15 were positive by the RCT. The increased sensitivity of the trivalent assay may have been due to the presence of large amounts of conserved and/or variable regions of the r56-kD protein from the three strains of O. tsutsugamushi. This may explain the high titers found in this assay compared with the whole cell and monovalent ELISAs (Figure 1). The benefit of this assay over that of the whole cell trivalent assay may be the presence of exposed binding sites on the recombinant proteins that may be hidden by other cell constituents in the whole cell preparation. We do not know whether the epitopes that were recognized by antibodies developed in scrub typhus patients had been induced by infection with more than one strain of O. tsutsugamushi. The variability of reactivity to the three recombinant proteins seen in the Western blot analysis confirms the importance of using antigens from multiple strains of O. tsutsugamushi in development of diagnostic assays for scrub typhus.
In conclusion, we were able to confirm the diagnosis of scrub typhus in two fever outbreaks that occurred among U.S. Marines training at Camp Fuji, Japan during the fall of 2000 and 2001 by using a new trivalent ELISA to detect antibodies directed against O. tsutsugamushi recombinant proteins (KpKtGm r56). Furthermore, this trivalent ELISA appears to be superior to the KpKtGm-wc ELISA and the Kp r56 ELISA, as well as the Kp r 56 RCT. This new ELISA may therefore provide a prototype for an assay to replace the whole cell and single recombinant antigen ELISAs in the laboratory. However, if one considers performance and ease of use, the RCT should have a place in the diagnosis of scrub typhus, especially in endemic areas where limited laboratory facilities are available and low numbers of specimens are being tested.
Onset and main symptoms in 19 suspected cases of scrub typhus among Marines deployed to Camp Fuji, Japan in 2000 and 2001*
| No. | Date first seen at base | Date of onset of symptoms | Fever, °C† | Rash | Eschar | Acute blood results‡ | Date convalescent blood collected |
|---|---|---|---|---|---|---|---|
| * ND = not determined. | |||||||
| † The maximum temperature recorded during the period of symptoms. | |||||||
| ‡ Indirect fluorescent antibody assay conducted at the Biomedical Laboratory in Tokyo, Japan. § 1:20 IgM and 1:10 IgG Gilliam, 1:20 IgM and <1:10 IgG Kato, and <1:10 IgM and IgG Karp. | |||||||
| ¶ 1:10 IgM Gilliam; the rest of the titers were < 1:10. | |||||||
| # 1:10 IgM and IgG Gilliam, 1:10 IgM and <1:10 IgG Kato, and <1:10 IgM and IgG Karp. | |||||||
| ** 1:40 IgM and IgG Gilliam, 1:20 IgM and IgG Kato, and 1:10 IgM and 1:20 IgG Karp. | |||||||
| 2001 | |||||||
| 1 | Oct 25 | Oct 25 | 38.2 | + | + | 1:20§ | Nov 29 |
| 2 | Oct 29 | Oct 27 | 38.8 | + | + | 1:10¶ | Nov 29 |
| 3 | Oct 27 | Oct 20 | 38.9 | + | + | 1:10# | Nov 29 |
| 4 | Oct 26 | Oct 26 | 38.2 | + | + | ND | Nov 29 |
| 5 | Oct 31 | Oct 29 | 37.7 | + | + | <1:10 | Nov 29 |
| 6 | Oct 31 | Oct 29 | 37.7 | + | “Mosquito bite” | <1:10 | Nov 29 |
| 7 | Nov 2 | Oct 28 | 38 | + | + | <1:10 | Nov 29 |
| 8 | Nov 3 | Oct 30 | 37.8 | + | “Macular lesion” | <1:10 | Nov 29 |
| 9 | Oct 27 | Oct 25 | 37.9 | + | + | 1:40** | Dec 6 |
| 10 | Oct 30 | Oct 28 | Reported | + | + | <1:10 | ND |
| 2002 | |||||||
| 1 | Nov 18 | Nov 11 | 37.4 | − | + | ND | Feb 27 |
| 2 | Nov 11 | Nov 1 | 37.0 | + | − | ND | Feb 27 |
| 3 | Nov 11 | Nov 5 | 37.4 | + | + | ND | Feb 27 |
| 4 | Nov 11 | Nov 7 | 39.4 | + | − | ND | Feb 27 |
| 5 | Nov 11 | Nov 6 | 38.6 | + | + | ND | Feb 27 |
| 6 | Nov 11 | Nov 6 | 38.4 | + | + | ND | Feb 27 |
| 7 | Nov 11 | Nov 5 | 36.4 | + | − | ND | Feb 27 |
| 8 | Nov 6 | Nov 2 | 38.9 | + | + | ND | Feb 27 |
| 9 | Nov 11 | Nov 4 | 37.6 | + | + | ND | Mar 6 |
Results of serologic tests conducted with convalescent sera collected from individuals involved in the 2000 scrub typhus outbreak at Camp Fuji, Japan*
| ELISA titer | RCT Kp r56 | Western blot IgG | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Group | No. | Whole cell | Kp r 56 | KpKtGm r56 | IgM | IgG | Kp r56 | Kt r56 | Gm r56 |
| * ELISA = enzyme-linked immunosorbent assay; RCT = cassette format rapid lateral flow assay; Kp = Karp; r56 = recombinant 56-kD proteins; Kt = Kato; Gm = Gilliam; ND = not determined. | |||||||||
| Suspect case | 1 | 400 | <100 | 1,600 | + | + | + | + | ++ |
| Suspect case | 2 | 1,600 | <100 | 400 | + | + | +/− | + | + |
| Suspect case | 3 | 1,600 | 6,400 | 6,400 | + | + | +++ | ++ | ++ |
| Suspect case | 4 | 100 | <100 | 6,400 | + | + | + | +/− | +++ |
| Suspect case | 5 | 400 | 1,600 | 1,600 | + | + | + | ++ | ++ |
| Suspect case | 6 | <100 | <100 | 400 | + | + | − | − | + |
| Suspect case | 7 | 400 | 6,400 | 6,400 | + | + | ++ | ++ | ++ |
| Suspect case | 8 | <100 | <100 | 1,600 | + | + | + | + | + |
| Suspect case | 9 | <100 | 100 | 1,600 | + | + | + | − | + |
| Suspect case | 10 | ND | ND | ND | ND | ND | ND | ND | ND |
| Control | 1 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 2 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 3 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 4 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 5 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 6 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 7 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 8 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 9 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 10 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 11 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 12 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 13 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 14 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 15 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 16 | <100 | <100 | <100 | − | − | ND | ND | ND |
| Control | 17 | <100 | <100 | <100 | − | − | ND | ND | ND |
Results of serologic tests conducted with sera collected from individuals involved in the 2001 scrub typhus outbreak at Camp Fuji, Japan*
Comparison of IgG antibody titers against Orientia tsutsugamushi determined by three enzyme-linked immunosorbent assays (ELISAs) (KpKtGm-wc ELISA, KpKtGm r56 ELISA, and Kp r56 ELISA) in sera collected from suspected cases in at Camp Fuji, Japan in 2000 and 2001. Kp = Karp; r56 = recombinant 56-kD protein; Kt = Kato; Gm = Gilliam; wc = whole cell.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 1; 10.4269/ajtmh.2003.69.60
Comparison of IgG antibody titers against Orientia tsutsugamushi determined by three enzyme-linked immunosorbent assays (ELISAs) (KpKtGm-wc ELISA, KpKtGm r56 ELISA, and Kp r56 ELISA) in sera collected from suspected cases in at Camp Fuji, Japan in 2000 and 2001. Kp = Karp; r56 = recombinant 56-kD protein; Kt = Kato; Gm = Gilliam; wc = whole cell.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 1; 10.4269/ajtmh.2003.69.60
Comparison of IgG antibody titers against Orientia tsutsugamushi determined by three enzyme-linked immunosorbent assays (ELISAs) (KpKtGm-wc ELISA, KpKtGm r56 ELISA, and Kp r56 ELISA) in sera collected from suspected cases in at Camp Fuji, Japan in 2000 and 2001. Kp = Karp; r56 = recombinant 56-kD protein; Kt = Kato; Gm = Gilliam; wc = whole cell.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 1; 10.4269/ajtmh.2003.69.60
Authors’ addresses: Ju Jiang, Rickettsial Diseases Department, Naval Medical Research Center 503 Robert Grant Avenue, Room 3N66, Silver Spring, MD 20910-7500, Telephone: 301-319-7249. Karen J. Marienau, Navy Environmental and Preventive Medicine Unit 6, 1215 North Road, Pearl Harbor, HI 96860-4477, Telephone: 808-473-0555, Fax: 808-473-2754. Laurel A. May, Navy Medical Clinic, 480 Central Avenue, Pearl Harbor, HI 96860-4477. H. James Beecham III, Naval Medical Research Unit No. 2, American Embassy, Jakarta, Indonesia FPO AP 96520-8132, Telephone: 62-21-425-6966, Fax: 62-21-424-4507. Ray Wilkinson, PANBIO Limited, 116 Lutwyche Road, Windsor, Brisbane, Queensland 4030, Australia, Telephone: 61-7-3357-1177, Fax: 61-7-3357-1222. Wei-Mei Ching, Rickettsial Diseases Department, Naval Medical Research Center 503 Robert Grant Avenue, Room 3N85, Silver Spring, MD 20910-7500, Telephone: 301-319-7438. Allen L. Richards, Rickettsial Diseases Department, Naval Medical Research Center 503 Robert Grant Avenue, Room 3A19, Silver Spring, MD 20910-7500.
Acknowledgments: We thank Dr. Shaun Peterson (3rd Battalion/3rd Marine Infantry Division, Marine Corps Base, Hawaii); Hospital Corpsman First Class Petty Officer Marcos Bordonado (3rd Battalion/3rd Marine Infantry Division, Marine Corps Base, Hawaii); and Chief Hospital Corpsman Bonnie D. O’Mara (Preventive Medicine Department, Naval Hospital, Yokosuka, Japan) for help during the 2000 Outbreak Investigation, and Hospital Corpsman First Class Petty Officer Andrew Barker; Hospital Corpsman Second Class Petty Officer Richard Cabitet; Hospital Corpsman Third Class Petty Officer Alvin Jules (1st Battalion/3rd Marine Infantry Division, Marine Corps Base, Hawaii); Lieutenant Alfredo Fernandez; Chief Hospital Corpsman Bonnie D. O’Mara (Preventive Medicine Department, Naval Hospital, Yokosuka, Japan); and Lieutenant Debra Baker (Laboratory Services, Naval Hospital, Okinawa Japan) for help during the 2001 Outbreak Investigation.
Financial support: This study was supported by Naval Medical Research Center (Work Unit no. 61102AS13JA006).
Disclaimer: The opinion and assertions contained herein are those of the authors and are not to be construed as official of reflecting the views of the U.S. Navy or the Department of Defense.
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