THE JOURNAL OF INFECTIOUS DISEASES • VOL. 134, NO.3' © 1976 by the University of Chicago. All rights reserved.
SEPTEMBER 1976
Prompt Confirmation of Rocky Mountain Spotted Fever: Identification of Rickettsiae in Skin Tissues T. E. Woodward, C. E. Pedersen, Jr., C. N. Oster, L. R. Bagley, J. Romberger, and M. J. Snyder
From the Department of Medicine, University of Maryland School of Medicine and Hospital, Baltimore; and the U.S. Army Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland
Many acute specific infections are similar in symptomatology during the early febrile days with such common manifestations as rapid onset, chills or chilly sensations, fever, prostration, malaise, myalgia, and headache. After several days, other characteristic features enable better determination by the physician of the most likely cause. With the rickettsioses, particularly Rocky Mountain spotted fever, which are prevalent throughout the United States, occurrence during the spring and summer, exposure in a rural or wooded section, and a tick bite as parts of the epidemiologic history are helpful clues. Yet, the physician may miss certain clues and mistake the headache, fever, myalgia, and rash of tick-borne spotted fever for measles, other viral illnesses, or meningococcemia. These errors, which can lead to delay in treatment, are evidenced by mortality rates of about 7 % in patients with spotted fever in the United States. Available confirmatory laboratory aids for rickettsial diseases include the Weil-Felix, CF, rickettsial micro agglutination (MA), and indirect fluorescent antibody (IFA) reactions, and the more tedious isolation of causative rickettsiae in animals or tissue culture. Regardless of the laboraReceived for publication July 6, 1976, and in revised form July 14, 1976. We thank. Dr. William C. Hall of the U.S. Army Research Institute of Infectious Diseases for preparation and interpretation of the photomicrograph. Please address requests for reprints to Dr. T. E. Woodward, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201.
tory method chosen, results are available late in illness when serious or irreversible vascular changes or death may have occurred. Recent attempts at earlier diagnosis have focused upon more rapid identification of the rickettsiae either by culture and specific identification of rickettsiae in monocytes from the buffy coat of the patient's blood or by identification of rickettsiae in vascular endothelium and other tissues by use of direct and indirect immunofluorescence [1, 2]. With these procedures Rickettsia rickettsii have been identified in monocytes and tissues of experimentally infected monkeys. We report rapid identification of R. rickettsii in skin specimens obtained by biopsy from patients with Rocky Mountain spotted fever as early as day 4 or as late as day 8 of illness. Materials and Methods
Patient selection and biopsy procedure. Physicians in Baltimore and other parts of Maryland referred patients with suspected Rocky Mountain spotted fever and helped to obtain consent for performance of skin biopsies. A full-thickness section of skin was removed by scalpel after thorough cleansing and local anesthesia. The specimen was placed in 0.85% NaCI and transported on ice. The small wound was sutured with silk. Skin specimens were obtained from patients with an early and late rash as well as from normal controls. The antisera used in this study and the preparation of conjugate have been described previously [2]. 297
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
Rickettsia rickettsii were identifiable by the immunofluorescence technique in skin specimens obtained by biopsy on days 4 and 8 of illness from patients with Rocky Mountain spotted fever. The immunofluorescence technique is regarded as a practical means of confirming the diagnosis during the early stages of illness before positive serologic reactions can be obtained.
298
on pressure were observed on the forearms, palms, legs, thighs, buttocks, and soles. A small section of skin from a fresh, pink macule was removed between the third and fourth days of illness. The patient's blood leukocyte count was 7,000/ mm", Values for serum sodium, chlorides, urea nitrogen, and protein were normal. Chloramphenicol treatment instituted on day 3 of illness was foIIowed by rapid improvement and defervescence in three days. The Proteus OX-19 (Weil-Felix), MA, and IFA reactions showed rises in titers from negative on day 3 of illness to 1: 64, 1: 256, and 1: 1,280 on day 28, respectively. The skin lesion obtained on the fourth febrile day was positive for the presence of rickettsiae by the indirect immunofluorescence test (figure 1). Case no. 2, an Ll-year-old Caucasian girl (V.C.W.) who lives in a wooded area near Libertytown, Md., was hospitalized at the Frederick Memorial Hospital on June 13, 1976, under the care of Dr. Charles Wright. Headache, which began on June 7, worsened. A pink rash appeared on about day 5 of illness and was assumed to be measles by the mother. Mental confusion developed on June 11 (about day 7 of illness). When hospitalized, the patient was in mild shock with muttering delirium. The face, arms, and legs were edematous, and the neck was moderately rigid; a diffuse, petechial, fixed rash involving the feet, legs, palms, soles, and forearms had begun to coalesce with purplish mottling and ecchymoses of the thighs. The trunk was involved to a lesser extent. Other signs were temperature of 105.7 F, pulse of 160, blood pressure of 90/60, clear lungs, cardiac sounds of poor quality, and no abdominal organomegaly or adenopathy. Laboratory findings included the following levels: blood leukocyte count, 10,300/mm3 ; serum sodium, 129 mEq/liter; chlorides, 92 mEq/liter; urea nitrogen, 26 mg/l00 ml; platelets, 87,000/mm3 ; and fibrinogen, 175 mg/100 mI. Treatment with chloramphenicol was begun on June 13 and with Solucortef (hydrocortisone) on the next day. The child was noticeably improved after two days of specific and supportive measures and became afebrile on day 6 of treatment. The titers in the Proteus OX-19, MA, and
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
Preparation of sections and staining. Frozen tissue sections (4 urn) were cut in a Cryostat II microtome (Ames Co., Elkhart, Ind.), mounted on thin, clean microscope slides, and fixed in cold (-70 C) acetone. Slides were stored at - 70 C until examined. For direct fluorescence microscopy, fluorescein-labeled guinea pig antiserum to R. rickettsii (titer, 1:2,560 by the fluorescent antibody reaction) was used at a working dilution of 1:30. Sections were soaked for 10 min in phosphate-buffered saline (PBS) , flooded for 30 min with the conjugated antiserum, and then rinsed for 6 min with PBS. For indirect immunofluorescence microscopy, sections were soaked for 10 min in PBS, dried with bibulous paper, and placed in a moist staining chamber. Sections were then flooded with guinea pig antiserum to R. rickettsii (diluted 1:50 in PBS, pH 7.3) for 45 min, washed for 30 min in PBS with gentle agitation, and blotted dry. In a moist staining chamber, slides were then flooded with fluorescein isothiocyanate-conjugated rabbit antiserum to guinea pig antigen (diluted 1:38 in PBS) for 45 min and washed with gentle agitation for 30 min in PBS. Cover slips were mounted with phosphate-buffered glycerol (nine parts of glycerol to one part of PBS) medium. Sections were examined with a Zeiss microscope equipped with a fluorescent illuminating system as previously described [2]. Histopathology. Tissues were fixed in neutral phosphate-buffered 10% formalin, processed through paraffin, and sectioned. Representative sections were stained with hematoxylin and eosin, Gimenez stain, and Giemsa stain [3]. Case reports. Case no. 1 was a four-year-old Caucasian girl (D.D.) who developed headache, fever, and anorexia on May 11, 1976; these symptoms persisted for several days. Pink skin lesions were noted on the arms on about day 3, and the eyes were reddened. The child was hospitalized at the Union Memorial Hospital on May 13, 1976, under the care of Dr. David Wood. Several ticks had been removed from the child during the previous weeks. On examination, she was fretful; her eyes were suffused and conjunctivae were congested. Findings of the lungs, heart (except for tachycardia), abdomen, and neurological and glandular systems were normal. Pink, slightly raised, irregular lesions that faded
Woodward et al.
Rickettsiae in Skin Tissue
299
Figure 1. Immunofluorescent preparation of Rickettsia rickettsii in a skin section from a macule taken on day 4 of Rocky Mountain spotted fever (case no. 1) . Note coccal and bacillary forms (X500).
IFA reactions on about day 8 of illness were 1 :80, 1:2, and 1:60, respectively, and 1:320, 1 :512, and 1:2,560 on day 23, respectively. The skin lesion taken on day 8 of illness was positive when examined by indirect immunofluorescence. The section of skin shown in figure 2 was stained with hematoxylin and eosin and shows the typical vasculitis of Rocky Mountain spotted fever . Full recovery ensued after convalescence which extended over several weeks. Results
Clinical findings. Each patient recovered with-. out sequelae, although patient no. 2, who was in mild shock with rickettsial encephalitis, required six days for defervescence and three weeks for full recovery. Laboratory diagnosis. Serology and confirmation of the agent. The specific diagnosis in each patient was confirmed by demonstration of diagnostic titers in the Weil-Felix, MA, and IFA tests. Skin biopsy for detection of rickettsiae by immunofluorescence. The cell (probably mononuclear) in figure 1 is from a specimen of a
Figure 2. Section of a hemorrhagic skin lesion taken on about day 8 of illness (case no. 2). There is segmented necrosis of dermal vessels and perivasular lymphoreticular infiltration (hematoxylin and eosin, X 290). The immunofluorescent preparation showed identifiable Rickettsia rickettsii.
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
macular skin lesion that was obtained on about day 4 of illness; rickettsiae showing bacillary and coccobacillary forms are readily distinguishable. Other sections also revealed easily distinguishable rickettsiae. Microscopy showed greenish-yellowtinted microorganisms. The control, normal skin specimen gave a negative result in this test, as did sections from the patient that were stained with fluorescein-conjugated normal serum globulin. Figure 2 is a hematoxylin and eosin-stained section of skin obtained from patient no. 2 on about day 8 of illness showing the microscopic lesions of Rocky Mountain spotted fever, segmental vascular necrosis including thrombosis within the arterioles, and perivascular exudate
300
Discussion
Ricketts, in a preliminary publication, reported the presence of "diplococcoid" microorganisms in the blood of patients with Rocky Mountain spotted fever and in concentrated serum of experimentally infected guinea pigs [4]. In pioneering studies from 1906 to 1909, Ricketts reported infectivity of guinea pigs and monkeys for the agent of Rocky Mountain spotted fever, transmission of infection to animals by the wood tick (Dermacentor andersoni), identification and life cycle of the microorganism in ticks, and important studies on immunity. Later, in classic studies of etiology, pathogenesis, and histopathology of Rocky Mountain spotted fever, Wolbach described rickettsiae in endothelial cells of vessels from tissues obtained postmortem, particularly in the locale of vascular lesions of scrotal skin, subcutaneous tissues, and skin associated with the exanthem [5]. Much later in 1950, Coons described the fluorescent antibody technique, which made it possible to identify epidemic typhus rickettsiae in the human body louse [6]. In 1960 and 1961, Burgdorfer used fluorescein isothiocyanate-Iabeled antibodies to identify R. rickettsii in tissues of infected ticks and guinea pigs [7, 8]. Various attempts have failed to confirm the clinical diagnosis of Rocky Mountain spotted fever during the early stages. In the 1930s, skin biopsy specimens stained by the Giemsa method showed in mast cells granules which were mistaken for rickettsiae. This procedure was abandoned. A serologic test in which rickettsial antigen obtained from the patient was used as the unknown against
standard antisera in a reverse CF reaction was unreliable. The physician must act upon clinical suspicion and judgment and on this basis institute specific treatment. Confirmatory information from serologic reactions accrues after the patient is well or in serious difficulty because of a delay in diagnosis and institution of specific treatment. A mortality rate of 7 % in a curable specific infection is unacceptable. With the immunofluorescence technique, it is now possible to localize rickettsiae in small specimens of skin obtained from the pink macular lesions as early as day 3 or 4 of illness or as late as day 8. Conceivably, these ranges may be extended. The time necessary to identify causative rickettsiae in the laboratory can be as short as 4 hr. A small skin specimen is placed in sterile saline and is processed promptly. (It may be frozen at -20 C to -70 C and examined later.) High-titered antiserum conjugated with fluorescein isothiocyanate is placed on properly sectioned skin specimens, which are then examined by darkfield microscopy. The usual controls are utilized. Rickettsiae have an identifiable morphology and characteristic staining properties. It may be possible to develop a more simplified technique, with use of a punch skin biopsy or needle aspiration of the purpuric lesion. Such a technique should be adaptable to other rickettsial diseases and certain specific infections associated with a rash. References 1. DeShazo, R. D., Boyce, J. R., Osterman, J. V., Stephenson, E. H. Early diagnosis of Rocky Mountain spotted fever. Use of primary monocyte culture technique. J.AM.A 235: 1353-1355, 1976. 2. Pedersen, C. E., Jr., Bagley, L. R., Kenyon, R. H., Sammons, L. S., Burger, G. T. Demonstration of Rickettsia rickettsii in the Rhesus monkey by immune fluorescence microscopy. J. Clin. MicrobioI. 2: 121-125, 1975. 3. Luna, L. G. [ed.]. Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. McGraw-Hill, New York, 1968. 258 p. 4. Ricketts, H. T. A micro-organism which apparently has a specific relationship to Rocky Mountain spotted fever. A preliminary report. J.AM.A. 5:379-380, 1909. 5. Wolbach, S. B. Studies on Rocky Mountain spotted fever. J. Med. Res. 41: 1-197, 1919.
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
mostly of lymphocytes and macrophages. Rickettsiae similar to those shown in figure 1 were identified in cells by the indirect immunofluorescence technique. There were fewer rickettsiae perhaps partly because of the influence of chloramphenicol, which had been given about 18 hr previously; conceivably, the host reaction resulted in fewer rickettsiae at this later stage of illness. Further studies are necessary to settle these points. Control skin specimens and sections of the patient's skin stained with normal serum did not show microorganisms.
Woodward et al.
Rickettsiae in Skin Tissue
6. Coons, A. H., Synder, J. C., Cheevers, F. S., Murray, E, S. Localization of antigen in tissue cells. IV. Antigens of rickettsiae and mumps virus. J. Exp. Med. 91 :31-37, 1950. 7. Burgdorfer, W., Lackman, D. Identification of Rickettsia rickettsii in the wood tick, Dermacentor
301
andersoni, by means of fluorescent antibody. J. Infect. Dis. 107:241-244, 1960. 8. Burgdorfer, W. Evaluation of the fluorescent antibody technique for the detection of Rocky Mountain spotted fever rickettsiae in various tissues. Pathol. Microbiol. (Basel) 24(Suppl.) :27-39, 1961.
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
SEPTEMBER 1976
Prompt Confirmation of Rocky Mountain Spotted Fever: Identification of Rickettsiae in Skin Tissues T. E. Woodward, C. E. Pedersen, Jr., C. N. Oster, L. R. Bagley, J. Romberger, and M. J. Snyder
From the Department of Medicine, University of Maryland School of Medicine and Hospital, Baltimore; and the U.S. Army Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland
Many acute specific infections are similar in symptomatology during the early febrile days with such common manifestations as rapid onset, chills or chilly sensations, fever, prostration, malaise, myalgia, and headache. After several days, other characteristic features enable better determination by the physician of the most likely cause. With the rickettsioses, particularly Rocky Mountain spotted fever, which are prevalent throughout the United States, occurrence during the spring and summer, exposure in a rural or wooded section, and a tick bite as parts of the epidemiologic history are helpful clues. Yet, the physician may miss certain clues and mistake the headache, fever, myalgia, and rash of tick-borne spotted fever for measles, other viral illnesses, or meningococcemia. These errors, which can lead to delay in treatment, are evidenced by mortality rates of about 7 % in patients with spotted fever in the United States. Available confirmatory laboratory aids for rickettsial diseases include the Weil-Felix, CF, rickettsial micro agglutination (MA), and indirect fluorescent antibody (IFA) reactions, and the more tedious isolation of causative rickettsiae in animals or tissue culture. Regardless of the laboraReceived for publication July 6, 1976, and in revised form July 14, 1976. We thank. Dr. William C. Hall of the U.S. Army Research Institute of Infectious Diseases for preparation and interpretation of the photomicrograph. Please address requests for reprints to Dr. T. E. Woodward, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201.
tory method chosen, results are available late in illness when serious or irreversible vascular changes or death may have occurred. Recent attempts at earlier diagnosis have focused upon more rapid identification of the rickettsiae either by culture and specific identification of rickettsiae in monocytes from the buffy coat of the patient's blood or by identification of rickettsiae in vascular endothelium and other tissues by use of direct and indirect immunofluorescence [1, 2]. With these procedures Rickettsia rickettsii have been identified in monocytes and tissues of experimentally infected monkeys. We report rapid identification of R. rickettsii in skin specimens obtained by biopsy from patients with Rocky Mountain spotted fever as early as day 4 or as late as day 8 of illness. Materials and Methods
Patient selection and biopsy procedure. Physicians in Baltimore and other parts of Maryland referred patients with suspected Rocky Mountain spotted fever and helped to obtain consent for performance of skin biopsies. A full-thickness section of skin was removed by scalpel after thorough cleansing and local anesthesia. The specimen was placed in 0.85% NaCI and transported on ice. The small wound was sutured with silk. Skin specimens were obtained from patients with an early and late rash as well as from normal controls. The antisera used in this study and the preparation of conjugate have been described previously [2]. 297
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
Rickettsia rickettsii were identifiable by the immunofluorescence technique in skin specimens obtained by biopsy on days 4 and 8 of illness from patients with Rocky Mountain spotted fever. The immunofluorescence technique is regarded as a practical means of confirming the diagnosis during the early stages of illness before positive serologic reactions can be obtained.
298
on pressure were observed on the forearms, palms, legs, thighs, buttocks, and soles. A small section of skin from a fresh, pink macule was removed between the third and fourth days of illness. The patient's blood leukocyte count was 7,000/ mm", Values for serum sodium, chlorides, urea nitrogen, and protein were normal. Chloramphenicol treatment instituted on day 3 of illness was foIIowed by rapid improvement and defervescence in three days. The Proteus OX-19 (Weil-Felix), MA, and IFA reactions showed rises in titers from negative on day 3 of illness to 1: 64, 1: 256, and 1: 1,280 on day 28, respectively. The skin lesion obtained on the fourth febrile day was positive for the presence of rickettsiae by the indirect immunofluorescence test (figure 1). Case no. 2, an Ll-year-old Caucasian girl (V.C.W.) who lives in a wooded area near Libertytown, Md., was hospitalized at the Frederick Memorial Hospital on June 13, 1976, under the care of Dr. Charles Wright. Headache, which began on June 7, worsened. A pink rash appeared on about day 5 of illness and was assumed to be measles by the mother. Mental confusion developed on June 11 (about day 7 of illness). When hospitalized, the patient was in mild shock with muttering delirium. The face, arms, and legs were edematous, and the neck was moderately rigid; a diffuse, petechial, fixed rash involving the feet, legs, palms, soles, and forearms had begun to coalesce with purplish mottling and ecchymoses of the thighs. The trunk was involved to a lesser extent. Other signs were temperature of 105.7 F, pulse of 160, blood pressure of 90/60, clear lungs, cardiac sounds of poor quality, and no abdominal organomegaly or adenopathy. Laboratory findings included the following levels: blood leukocyte count, 10,300/mm3 ; serum sodium, 129 mEq/liter; chlorides, 92 mEq/liter; urea nitrogen, 26 mg/l00 ml; platelets, 87,000/mm3 ; and fibrinogen, 175 mg/100 mI. Treatment with chloramphenicol was begun on June 13 and with Solucortef (hydrocortisone) on the next day. The child was noticeably improved after two days of specific and supportive measures and became afebrile on day 6 of treatment. The titers in the Proteus OX-19, MA, and
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
Preparation of sections and staining. Frozen tissue sections (4 urn) were cut in a Cryostat II microtome (Ames Co., Elkhart, Ind.), mounted on thin, clean microscope slides, and fixed in cold (-70 C) acetone. Slides were stored at - 70 C until examined. For direct fluorescence microscopy, fluorescein-labeled guinea pig antiserum to R. rickettsii (titer, 1:2,560 by the fluorescent antibody reaction) was used at a working dilution of 1:30. Sections were soaked for 10 min in phosphate-buffered saline (PBS) , flooded for 30 min with the conjugated antiserum, and then rinsed for 6 min with PBS. For indirect immunofluorescence microscopy, sections were soaked for 10 min in PBS, dried with bibulous paper, and placed in a moist staining chamber. Sections were then flooded with guinea pig antiserum to R. rickettsii (diluted 1:50 in PBS, pH 7.3) for 45 min, washed for 30 min in PBS with gentle agitation, and blotted dry. In a moist staining chamber, slides were then flooded with fluorescein isothiocyanate-conjugated rabbit antiserum to guinea pig antigen (diluted 1:38 in PBS) for 45 min and washed with gentle agitation for 30 min in PBS. Cover slips were mounted with phosphate-buffered glycerol (nine parts of glycerol to one part of PBS) medium. Sections were examined with a Zeiss microscope equipped with a fluorescent illuminating system as previously described [2]. Histopathology. Tissues were fixed in neutral phosphate-buffered 10% formalin, processed through paraffin, and sectioned. Representative sections were stained with hematoxylin and eosin, Gimenez stain, and Giemsa stain [3]. Case reports. Case no. 1 was a four-year-old Caucasian girl (D.D.) who developed headache, fever, and anorexia on May 11, 1976; these symptoms persisted for several days. Pink skin lesions were noted on the arms on about day 3, and the eyes were reddened. The child was hospitalized at the Union Memorial Hospital on May 13, 1976, under the care of Dr. David Wood. Several ticks had been removed from the child during the previous weeks. On examination, she was fretful; her eyes were suffused and conjunctivae were congested. Findings of the lungs, heart (except for tachycardia), abdomen, and neurological and glandular systems were normal. Pink, slightly raised, irregular lesions that faded
Woodward et al.
Rickettsiae in Skin Tissue
299
Figure 1. Immunofluorescent preparation of Rickettsia rickettsii in a skin section from a macule taken on day 4 of Rocky Mountain spotted fever (case no. 1) . Note coccal and bacillary forms (X500).
IFA reactions on about day 8 of illness were 1 :80, 1:2, and 1:60, respectively, and 1:320, 1 :512, and 1:2,560 on day 23, respectively. The skin lesion taken on day 8 of illness was positive when examined by indirect immunofluorescence. The section of skin shown in figure 2 was stained with hematoxylin and eosin and shows the typical vasculitis of Rocky Mountain spotted fever . Full recovery ensued after convalescence which extended over several weeks. Results
Clinical findings. Each patient recovered with-. out sequelae, although patient no. 2, who was in mild shock with rickettsial encephalitis, required six days for defervescence and three weeks for full recovery. Laboratory diagnosis. Serology and confirmation of the agent. The specific diagnosis in each patient was confirmed by demonstration of diagnostic titers in the Weil-Felix, MA, and IFA tests. Skin biopsy for detection of rickettsiae by immunofluorescence. The cell (probably mononuclear) in figure 1 is from a specimen of a
Figure 2. Section of a hemorrhagic skin lesion taken on about day 8 of illness (case no. 2). There is segmented necrosis of dermal vessels and perivasular lymphoreticular infiltration (hematoxylin and eosin, X 290). The immunofluorescent preparation showed identifiable Rickettsia rickettsii.
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
macular skin lesion that was obtained on about day 4 of illness; rickettsiae showing bacillary and coccobacillary forms are readily distinguishable. Other sections also revealed easily distinguishable rickettsiae. Microscopy showed greenish-yellowtinted microorganisms. The control, normal skin specimen gave a negative result in this test, as did sections from the patient that were stained with fluorescein-conjugated normal serum globulin. Figure 2 is a hematoxylin and eosin-stained section of skin obtained from patient no. 2 on about day 8 of illness showing the microscopic lesions of Rocky Mountain spotted fever, segmental vascular necrosis including thrombosis within the arterioles, and perivascular exudate
300
Discussion
Ricketts, in a preliminary publication, reported the presence of "diplococcoid" microorganisms in the blood of patients with Rocky Mountain spotted fever and in concentrated serum of experimentally infected guinea pigs [4]. In pioneering studies from 1906 to 1909, Ricketts reported infectivity of guinea pigs and monkeys for the agent of Rocky Mountain spotted fever, transmission of infection to animals by the wood tick (Dermacentor andersoni), identification and life cycle of the microorganism in ticks, and important studies on immunity. Later, in classic studies of etiology, pathogenesis, and histopathology of Rocky Mountain spotted fever, Wolbach described rickettsiae in endothelial cells of vessels from tissues obtained postmortem, particularly in the locale of vascular lesions of scrotal skin, subcutaneous tissues, and skin associated with the exanthem [5]. Much later in 1950, Coons described the fluorescent antibody technique, which made it possible to identify epidemic typhus rickettsiae in the human body louse [6]. In 1960 and 1961, Burgdorfer used fluorescein isothiocyanate-Iabeled antibodies to identify R. rickettsii in tissues of infected ticks and guinea pigs [7, 8]. Various attempts have failed to confirm the clinical diagnosis of Rocky Mountain spotted fever during the early stages. In the 1930s, skin biopsy specimens stained by the Giemsa method showed in mast cells granules which were mistaken for rickettsiae. This procedure was abandoned. A serologic test in which rickettsial antigen obtained from the patient was used as the unknown against
standard antisera in a reverse CF reaction was unreliable. The physician must act upon clinical suspicion and judgment and on this basis institute specific treatment. Confirmatory information from serologic reactions accrues after the patient is well or in serious difficulty because of a delay in diagnosis and institution of specific treatment. A mortality rate of 7 % in a curable specific infection is unacceptable. With the immunofluorescence technique, it is now possible to localize rickettsiae in small specimens of skin obtained from the pink macular lesions as early as day 3 or 4 of illness or as late as day 8. Conceivably, these ranges may be extended. The time necessary to identify causative rickettsiae in the laboratory can be as short as 4 hr. A small skin specimen is placed in sterile saline and is processed promptly. (It may be frozen at -20 C to -70 C and examined later.) High-titered antiserum conjugated with fluorescein isothiocyanate is placed on properly sectioned skin specimens, which are then examined by darkfield microscopy. The usual controls are utilized. Rickettsiae have an identifiable morphology and characteristic staining properties. It may be possible to develop a more simplified technique, with use of a punch skin biopsy or needle aspiration of the purpuric lesion. Such a technique should be adaptable to other rickettsial diseases and certain specific infections associated with a rash. References 1. DeShazo, R. D., Boyce, J. R., Osterman, J. V., Stephenson, E. H. Early diagnosis of Rocky Mountain spotted fever. Use of primary monocyte culture technique. J.AM.A 235: 1353-1355, 1976. 2. Pedersen, C. E., Jr., Bagley, L. R., Kenyon, R. H., Sammons, L. S., Burger, G. T. Demonstration of Rickettsia rickettsii in the Rhesus monkey by immune fluorescence microscopy. J. Clin. MicrobioI. 2: 121-125, 1975. 3. Luna, L. G. [ed.]. Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. McGraw-Hill, New York, 1968. 258 p. 4. Ricketts, H. T. A micro-organism which apparently has a specific relationship to Rocky Mountain spotted fever. A preliminary report. J.AM.A. 5:379-380, 1909. 5. Wolbach, S. B. Studies on Rocky Mountain spotted fever. J. Med. Res. 41: 1-197, 1919.
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
mostly of lymphocytes and macrophages. Rickettsiae similar to those shown in figure 1 were identified in cells by the indirect immunofluorescence technique. There were fewer rickettsiae perhaps partly because of the influence of chloramphenicol, which had been given about 18 hr previously; conceivably, the host reaction resulted in fewer rickettsiae at this later stage of illness. Further studies are necessary to settle these points. Control skin specimens and sections of the patient's skin stained with normal serum did not show microorganisms.
Woodward et al.
Rickettsiae in Skin Tissue
6. Coons, A. H., Synder, J. C., Cheevers, F. S., Murray, E, S. Localization of antigen in tissue cells. IV. Antigens of rickettsiae and mumps virus. J. Exp. Med. 91 :31-37, 1950. 7. Burgdorfer, W., Lackman, D. Identification of Rickettsia rickettsii in the wood tick, Dermacentor
301
andersoni, by means of fluorescent antibody. J. Infect. Dis. 107:241-244, 1960. 8. Burgdorfer, W. Evaluation of the fluorescent antibody technique for the detection of Rocky Mountain spotted fever rickettsiae in various tissues. Pathol. Microbiol. (Basel) 24(Suppl.) :27-39, 1961.
Downloaded from http://jid.oxfordjournals.org/ at University of Exeter on August 16, 2015
