THE JOURNAL OF INFECTIOUS DISEASES • VOL. 133, NO.5· © 1976 by the University of Chicago. All rights reserved.
MAY 1976
MAJOR ARTICLES Yersinia pestis Infection in Vietnam. II; Quantitative Blood Cultures and Detection of Endotoxin in the Cerebrospinal Fluid of Patients with Meningitis Thomas Butler, Jack Levin, Nguyen Ngoc Linh, * Duong Minh Chao, Michael Adickman, and Keith Arnold
Quantitative blood cultures were obtained from 42 patients with acute Yersinia pestis infection to determine whether the concentration of bacteria in blood influenced the clinical severity and outcome of illness. In 17 bacteremic patients, colony counts in blood cultures ranged from < 10 to 4 X 10 7 / ml. Three of five patients with colony counts of >102 / ml died, and two patients survived episodes of hypotension. Results from plasma limulus tests were positive at the time of admission in three of 10 patients tested, and these three patients had bacteremia with colony counts of > 102 / ml, Meningitis developed in three patients and pneumonia in two patients; these five patients all had buboes in the axillary region. Endotoxin was detected with the limulus test in the cerebrospinal fluid in the three patients with meningitis. Ten patients randomly assigned to receive streptomycin or trimethoprim-sulfamethoxazole survived. Those treated with streptomycin had a shorter median duration of fever and a lower incidence of complications than did the patients treated with trimethoprim-sulfamethoxazole.
A remarkable feature of infections caused by Yersinia pestis is the occasional presence of bacteria in the blood in such high concentration that they
are easily seen in peripheral blood smears [1, 2]. This feature appears to be associated with a high mortality rate and has been proposed as a useful prognostic sign in human plague [3, 4]. In 1952, Meyer et al. recommended semiquantitative blood cultures to determine the prognosis, since > 40 bacteria/rnl proved to be fatal in 90% of patients in India who were treated with sulfonaniides [5]. In the current study, patients who had concentrations of circulating bacteria of > 102/ml had more severe illnesses and a higher mortality rate than other patients. Endotoxin, as defined by the limulus test, is present in the blood of some patients acutely ill with plague [6]. The limulus test also has been shown to detect accurately endotoxin in the cerebrospinal fluid of patients with meningitis due to gram-negative organisms and to distinguish these patients from those with meningitis due to a wide variety of other causes [7]. In this study the cerebrospinal fluid of three patients with meningitis produced positive results in the limulus test.
Received for publication October 24, 1974, and in revised form December 9, 1975. These studies were supported in part by grant no. RDRF S92-544-74-0212 from the Department of the Army; training grant no. AI 00009 from the National Institutes of Health; research grant no. HL 01601 from the National Heart and Lung Institute; Roche (Far East); and a grant from the Rockefeller Foundation. Dr. Levin is the recipient of a Research Career Development Award (no. K04 HL 29906) from the National Heart and Lung Institute. We are grateful for the assistance of Vu thi Dan, Do thi Minh Tam, Mach Muoi Muoi, Amalia Franco, Francine Corthesy, and Tran Kim Anh; for the laboratory support of Nguyen Huu Tu, Dan Cavanaugh, Daniel Harrison, and Bruce Hudson; and for the statistical advice of Susan Horn. Please address requests for reprints to Dr. Thomas Butler, Division of Geographic Medicine, Department of Medicine, University Hospitals, Cleveland, Ohio 44106. * Deceased, April 1975. 493
Downloaded from http://jid.oxfordjournals.org/ at Stanford Medical Center on October 12, 2012
From the Cho-Quan Hospital, Saigon, South Vietnam; the Divisions of Infectious Diseases and Hematology, Department of Medicine, Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland,' the Division of Geographic Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio; and the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
Butler et al.
494
Materials and Methods
[11].
Specimens of cerebrospinal fluid for endotoxin assay were placed in pyrogen-free containers with penicillin G (final concentration, 100 units/ml) and streptomycin (final concentration, 100 ~g/ ml) and frozen. Blood for endotoxin assay was collected in pyrogen-free test tubes which contained heparin in a final concentration of 100 units/rnl. The acute-phase specimen was taken at the time of admission, and the convalescent-phase specimen was taken after 10 days of treatment or prior to discharge. Plasma was placed in tubes which contained penicillin G and streptomycin in
Results
Patients. There were 22 females and 20 males whose ages ranged from three to 70 years (median age, 15 years). In six instances there were clusters of two or three patients in the same family who were in the hospital at the same time. An apparent peak of the epidemic occurred in the latter part of March and early part of April 1974. Twenty-three patients lived in the well-known plague focus of CuChi in Hau Nghia Province, seven patients came from nearby districts in Hau Nghia Province, 11 patients were from the neighboring province of Tay Ninh, and one was from Long Thanh. Clinical features. Most patients presented with the abrupt onset of fever, chills, and painful buboes. The duration of symptoms prior to admission ranged from one to seven days (median, three days) . Twenty-four patients appeared acutely toxic with altered mental status including
Downloaded from http://jid.oxfordjournals.org/ at Stanford Medical Center on October 12, 2012
From February 1974 to April 1975, 42 patients admitted to the Cho-Quan Hospital in Saigon with acute adenitis were demonstrated to have Y. pestis infection by positive cultures of bubo aspirate, blood, or cerebrospinal fluid or by a rise in titer of convalescent-phase antibody to Fraction I antigen of Y. pestis [8]. Material for culture was injected into Vacutainer® tubes (Becton-Dickinson, Rutherford, N.J.) containing supplemental peptone broth with penicillinase. Y. pestis was identified by microscopic morphology, biochemical reactions, and specific bacteriophage lysis [9]. For quantitative blood cultures, 0.1 ml of undiluted blood and serial l Osfold dilutions of blood in 0.9% NaCI were streaked evenly onto deoxycholate agar plates. The number of characteristic colonies appearing on each plate was multiplied by the respective dilution factor to obtain the colony count in blood. The colony count was expressed as a power of 10 multiplied by the nearest integer per ml. No estimate of error was made because only single determinations were possible under our field conditions. All blood cultures were obtained prior to initiation of treatment in the hospital. Some patients had histories of possible antimicrobial treatment prior to admission, but previous treatment could not be documented in any case. Cultures were transported to Baltimore on Cary-Blair transport media. Antibiotic susceptibilities were tested by the Kirby-Bauer technique [10] on Mueller-Hinton agar. Susceptibilities to trimethoprim, sulfamethoxazole, and combinations of these drugs were determined by an agar dilution method with use of Mueller-Hinton agar containing 7.5% lysed horse blood and drugs
the same concentrations as those for cerebrospinal fluid. The limulus test for endotoxin was performed by incubation of 0.05 ml of plasma or cerebrospinal fluid with 0.05 ml of limulus amebocyte lysate as previously described [12, 13]. Specimens of cerebrospinal fluid that were positive were serially diluted in lOa-fold steps in 0.9% NaCI and retested. In this study, concentrations of endotoxin as low as 0.001 ug/rnl in plasma and 0.0001 ug/rnl in cerebrospinal fluid were detected by the limulus test with use of Escherichia coli endotoxin as a standard (Difco lipopolysaccharide, E. coli B, 055:B5; Difco, Detroit, Mich.). The 32 patients admitted in 1974 received streptomycin or streptomycin with chloramphenicol as chosen by the local physician. In 1975 the 10 patients were randomly allocated to receive either streptomycin or Bactrim® (160 mg of trimethoprim and 800 mg of sulfamethoxazole; Roche Laboratories, Basel, Switzerland). Streptomycin was given in an im dose of 1 g twice daily for 10 days, and patients weighing < 25 kg received half this dose. Bactrim® was given iv twice daily for three to five days, after which tablets with the same drug content were given to complete a l O-day course. Children < 12 years old received half this dose.
495
Yersinia pestis Infection in Vietnam
Table 1. Incidence of hypotension and death related to quantitative blood cultures in 42 patients with Yersinia pestis infection. Status (colony count) Bacteremic c- 102 colonies/ml) Bacteremic (1-10 2 colonies/ml) Not bacteremic
Total no. of patients
No. with hypotension *
No. of deaths
11
5
3
6 25
2
o
o
It
* Systolic pressure, ===: 80 mm Hg. t This patient had meningitis. mic and five who were not bacteremic, were tested for endotoxemia in their acute and convalescent stages of illness. Table 2 shows that results of limulus tests were positive during the acute stage of illness in three patients with colony counts of > 102/ml. These three patients had severe illnesses, including hypotension and renal failure. Two patients with positive test results during convalescence had longer febrile courses than the other patients, and one of these, no. 4, developed pneumonia. All 10 patients survived. Meningitis and detection of endotoxin in cerebrospinal fluid. Three patients had meningitis. All had symptoms of plague for six to 10 days prior to lumbar puncture, as well as axillary buboes and negative blood cultures. As shown in table 3, results of limulus tests performed on cerebrospinal fluid were positive. Patients no. M-1 and M-3 had negative cerebrospinal fluid cultures without bacteria, as tested by gram stain of the spinal fluid, but these patients had been treated for two and five days, respectively, with antimicrobial drugs prior to lumbar puncture. Patient no. M-2 had a positive cerebrospinal fluid culture and plentiful bacteria detected by gram stain. Therapy and hospital courses. All four deaths occurred in 1974 during the first 24 hr in the hospital despite antibiotic therapy. In 1974, 13 of the surviving patients were treated with streptomycin alone, and 15 were treated with streptomycin and chloramphenicol. In these survivors, the number of febrile days (including the day of admission) during which a rectal temperature of > 38 C was recorded ranged from one to 11 with a median of four days. In 1975, streptomycin was given to five patients, including no. 1 in table 2, who had fever for 16 days, and the median duration of fever was
Downloaded from http://jid.oxfordjournals.org/ at Stanford Medical Center on October 12, 2012
lethargy, stupor, and delirium; the remaining 18 patients had milder systemic symptoms and were alert. Buboes were tender in all cases and were located in the femoral region in 22 patients, axillary in 15 patients, inguinal in 11 patients, cervical in three patients, supraclavicular in one patient, and epitrochlear in one patient. Temperatures of patients at the time of admission ranged from 36.7 C to 41.5 C with a mean of 39.3 C. Hypotension (systolic blood pressure ~ 80 mm Hg) was present in seven patients. White blood cell counts ranged from 4,500 to 58,800/mm3 with a mean of 18,486/mm3 • Cultures and antimicrobial susceptibilities. Bubo aspirates were cultured in 41 patients at the time of admission and yielded Y. pestis in 35 cases. Blood cultures of 42 patients yielded Y. pestis in 17 cases; one of three cerebrospinal fluid cultures was positive, one pustule was positive, and three patients had multiple positive bubo aspirates. One patient who had negative cultures showed a diagnostic increase in titer of antibody to Fraction I of Y. pestis. Susceptibility testing by the Kirby-Bauer method revealed that all 56 of the tested isolates were susceptible to ampicillin, cephalothin, chloramphenicol, streptomycin, and tetracycline. For 45 isolates tested by agar dilution, the MIC of trimethoprim was 1 ug/rnl for 40 isolates, 0.3 ug/ml for three isolates, and 0.03 J.1g/ ml for two isolates. The MIC of sulfamethoxazole was 100J.1g/ml for 38 isolates, 30 ug/rnl for three isolates, and 10J.1g/ml for four isolates. When the drugs were combined, the MIC for 26 isolates was 0.1 ug of trimethoprim/rnl and 2 J.1g of sulfamethoxazole/ml; for 19 isolates the MIC was 0.03 J.1g of trimethoprim/ml and 0.6 J.1g of sulfamethoxazole/ ml. Blood cultures. Seventeen patients had bacteremia with colony counts ranging from < 10 to 4 X 107 colonies /ml. In four patients with colony counts of > 106/ml, bacteria were easily visible in the peripheral blood smears stained with Wright-Giemsa stain. Table 1 shows that 11 patients with colony counts of > 102/ml had higher incidences of hypotension and death than patients with lower colony counts and negative blood cultures. The patient who died without bacteremia had meningitis. Detection of endotoxemia. Ten patients studied in 1975, including five who were bactere-
Butler et al.
496
Table 2. Quantitative blood cultures, plasma limulus test results, and clinical features in 10 patients with Yersinia pestis infection. Plasma limulus test*
Quantitative blood culture (colonies/ml)
Acutephase specimen
Convalescentphase specimen
(21, F)
1 X 107
+
+
2
(28, F)
2 X 104
+
Femoral
3
(13, M)
Negative
4
(18, F)
MAY 1976
MAJOR ARTICLES Yersinia pestis Infection in Vietnam. II; Quantitative Blood Cultures and Detection of Endotoxin in the Cerebrospinal Fluid of Patients with Meningitis Thomas Butler, Jack Levin, Nguyen Ngoc Linh, * Duong Minh Chao, Michael Adickman, and Keith Arnold
Quantitative blood cultures were obtained from 42 patients with acute Yersinia pestis infection to determine whether the concentration of bacteria in blood influenced the clinical severity and outcome of illness. In 17 bacteremic patients, colony counts in blood cultures ranged from < 10 to 4 X 10 7 / ml. Three of five patients with colony counts of >102 / ml died, and two patients survived episodes of hypotension. Results from plasma limulus tests were positive at the time of admission in three of 10 patients tested, and these three patients had bacteremia with colony counts of > 102 / ml, Meningitis developed in three patients and pneumonia in two patients; these five patients all had buboes in the axillary region. Endotoxin was detected with the limulus test in the cerebrospinal fluid in the three patients with meningitis. Ten patients randomly assigned to receive streptomycin or trimethoprim-sulfamethoxazole survived. Those treated with streptomycin had a shorter median duration of fever and a lower incidence of complications than did the patients treated with trimethoprim-sulfamethoxazole.
A remarkable feature of infections caused by Yersinia pestis is the occasional presence of bacteria in the blood in such high concentration that they
are easily seen in peripheral blood smears [1, 2]. This feature appears to be associated with a high mortality rate and has been proposed as a useful prognostic sign in human plague [3, 4]. In 1952, Meyer et al. recommended semiquantitative blood cultures to determine the prognosis, since > 40 bacteria/rnl proved to be fatal in 90% of patients in India who were treated with sulfonaniides [5]. In the current study, patients who had concentrations of circulating bacteria of > 102/ml had more severe illnesses and a higher mortality rate than other patients. Endotoxin, as defined by the limulus test, is present in the blood of some patients acutely ill with plague [6]. The limulus test also has been shown to detect accurately endotoxin in the cerebrospinal fluid of patients with meningitis due to gram-negative organisms and to distinguish these patients from those with meningitis due to a wide variety of other causes [7]. In this study the cerebrospinal fluid of three patients with meningitis produced positive results in the limulus test.
Received for publication October 24, 1974, and in revised form December 9, 1975. These studies were supported in part by grant no. RDRF S92-544-74-0212 from the Department of the Army; training grant no. AI 00009 from the National Institutes of Health; research grant no. HL 01601 from the National Heart and Lung Institute; Roche (Far East); and a grant from the Rockefeller Foundation. Dr. Levin is the recipient of a Research Career Development Award (no. K04 HL 29906) from the National Heart and Lung Institute. We are grateful for the assistance of Vu thi Dan, Do thi Minh Tam, Mach Muoi Muoi, Amalia Franco, Francine Corthesy, and Tran Kim Anh; for the laboratory support of Nguyen Huu Tu, Dan Cavanaugh, Daniel Harrison, and Bruce Hudson; and for the statistical advice of Susan Horn. Please address requests for reprints to Dr. Thomas Butler, Division of Geographic Medicine, Department of Medicine, University Hospitals, Cleveland, Ohio 44106. * Deceased, April 1975. 493
Downloaded from http://jid.oxfordjournals.org/ at Stanford Medical Center on October 12, 2012
From the Cho-Quan Hospital, Saigon, South Vietnam; the Divisions of Infectious Diseases and Hematology, Department of Medicine, Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland,' the Division of Geographic Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio; and the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
Butler et al.
494
Materials and Methods
[11].
Specimens of cerebrospinal fluid for endotoxin assay were placed in pyrogen-free containers with penicillin G (final concentration, 100 units/ml) and streptomycin (final concentration, 100 ~g/ ml) and frozen. Blood for endotoxin assay was collected in pyrogen-free test tubes which contained heparin in a final concentration of 100 units/rnl. The acute-phase specimen was taken at the time of admission, and the convalescent-phase specimen was taken after 10 days of treatment or prior to discharge. Plasma was placed in tubes which contained penicillin G and streptomycin in
Results
Patients. There were 22 females and 20 males whose ages ranged from three to 70 years (median age, 15 years). In six instances there were clusters of two or three patients in the same family who were in the hospital at the same time. An apparent peak of the epidemic occurred in the latter part of March and early part of April 1974. Twenty-three patients lived in the well-known plague focus of CuChi in Hau Nghia Province, seven patients came from nearby districts in Hau Nghia Province, 11 patients were from the neighboring province of Tay Ninh, and one was from Long Thanh. Clinical features. Most patients presented with the abrupt onset of fever, chills, and painful buboes. The duration of symptoms prior to admission ranged from one to seven days (median, three days) . Twenty-four patients appeared acutely toxic with altered mental status including
Downloaded from http://jid.oxfordjournals.org/ at Stanford Medical Center on October 12, 2012
From February 1974 to April 1975, 42 patients admitted to the Cho-Quan Hospital in Saigon with acute adenitis were demonstrated to have Y. pestis infection by positive cultures of bubo aspirate, blood, or cerebrospinal fluid or by a rise in titer of convalescent-phase antibody to Fraction I antigen of Y. pestis [8]. Material for culture was injected into Vacutainer® tubes (Becton-Dickinson, Rutherford, N.J.) containing supplemental peptone broth with penicillinase. Y. pestis was identified by microscopic morphology, biochemical reactions, and specific bacteriophage lysis [9]. For quantitative blood cultures, 0.1 ml of undiluted blood and serial l Osfold dilutions of blood in 0.9% NaCI were streaked evenly onto deoxycholate agar plates. The number of characteristic colonies appearing on each plate was multiplied by the respective dilution factor to obtain the colony count in blood. The colony count was expressed as a power of 10 multiplied by the nearest integer per ml. No estimate of error was made because only single determinations were possible under our field conditions. All blood cultures were obtained prior to initiation of treatment in the hospital. Some patients had histories of possible antimicrobial treatment prior to admission, but previous treatment could not be documented in any case. Cultures were transported to Baltimore on Cary-Blair transport media. Antibiotic susceptibilities were tested by the Kirby-Bauer technique [10] on Mueller-Hinton agar. Susceptibilities to trimethoprim, sulfamethoxazole, and combinations of these drugs were determined by an agar dilution method with use of Mueller-Hinton agar containing 7.5% lysed horse blood and drugs
the same concentrations as those for cerebrospinal fluid. The limulus test for endotoxin was performed by incubation of 0.05 ml of plasma or cerebrospinal fluid with 0.05 ml of limulus amebocyte lysate as previously described [12, 13]. Specimens of cerebrospinal fluid that were positive were serially diluted in lOa-fold steps in 0.9% NaCI and retested. In this study, concentrations of endotoxin as low as 0.001 ug/rnl in plasma and 0.0001 ug/rnl in cerebrospinal fluid were detected by the limulus test with use of Escherichia coli endotoxin as a standard (Difco lipopolysaccharide, E. coli B, 055:B5; Difco, Detroit, Mich.). The 32 patients admitted in 1974 received streptomycin or streptomycin with chloramphenicol as chosen by the local physician. In 1975 the 10 patients were randomly allocated to receive either streptomycin or Bactrim® (160 mg of trimethoprim and 800 mg of sulfamethoxazole; Roche Laboratories, Basel, Switzerland). Streptomycin was given in an im dose of 1 g twice daily for 10 days, and patients weighing < 25 kg received half this dose. Bactrim® was given iv twice daily for three to five days, after which tablets with the same drug content were given to complete a l O-day course. Children < 12 years old received half this dose.
495
Yersinia pestis Infection in Vietnam
Table 1. Incidence of hypotension and death related to quantitative blood cultures in 42 patients with Yersinia pestis infection. Status (colony count) Bacteremic c- 102 colonies/ml) Bacteremic (1-10 2 colonies/ml) Not bacteremic
Total no. of patients
No. with hypotension *
No. of deaths
11
5
3
6 25
2
o
o
It
* Systolic pressure, ===: 80 mm Hg. t This patient had meningitis. mic and five who were not bacteremic, were tested for endotoxemia in their acute and convalescent stages of illness. Table 2 shows that results of limulus tests were positive during the acute stage of illness in three patients with colony counts of > 102/ml. These three patients had severe illnesses, including hypotension and renal failure. Two patients with positive test results during convalescence had longer febrile courses than the other patients, and one of these, no. 4, developed pneumonia. All 10 patients survived. Meningitis and detection of endotoxin in cerebrospinal fluid. Three patients had meningitis. All had symptoms of plague for six to 10 days prior to lumbar puncture, as well as axillary buboes and negative blood cultures. As shown in table 3, results of limulus tests performed on cerebrospinal fluid were positive. Patients no. M-1 and M-3 had negative cerebrospinal fluid cultures without bacteria, as tested by gram stain of the spinal fluid, but these patients had been treated for two and five days, respectively, with antimicrobial drugs prior to lumbar puncture. Patient no. M-2 had a positive cerebrospinal fluid culture and plentiful bacteria detected by gram stain. Therapy and hospital courses. All four deaths occurred in 1974 during the first 24 hr in the hospital despite antibiotic therapy. In 1974, 13 of the surviving patients were treated with streptomycin alone, and 15 were treated with streptomycin and chloramphenicol. In these survivors, the number of febrile days (including the day of admission) during which a rectal temperature of > 38 C was recorded ranged from one to 11 with a median of four days. In 1975, streptomycin was given to five patients, including no. 1 in table 2, who had fever for 16 days, and the median duration of fever was
Downloaded from http://jid.oxfordjournals.org/ at Stanford Medical Center on October 12, 2012
lethargy, stupor, and delirium; the remaining 18 patients had milder systemic symptoms and were alert. Buboes were tender in all cases and were located in the femoral region in 22 patients, axillary in 15 patients, inguinal in 11 patients, cervical in three patients, supraclavicular in one patient, and epitrochlear in one patient. Temperatures of patients at the time of admission ranged from 36.7 C to 41.5 C with a mean of 39.3 C. Hypotension (systolic blood pressure ~ 80 mm Hg) was present in seven patients. White blood cell counts ranged from 4,500 to 58,800/mm3 with a mean of 18,486/mm3 • Cultures and antimicrobial susceptibilities. Bubo aspirates were cultured in 41 patients at the time of admission and yielded Y. pestis in 35 cases. Blood cultures of 42 patients yielded Y. pestis in 17 cases; one of three cerebrospinal fluid cultures was positive, one pustule was positive, and three patients had multiple positive bubo aspirates. One patient who had negative cultures showed a diagnostic increase in titer of antibody to Fraction I of Y. pestis. Susceptibility testing by the Kirby-Bauer method revealed that all 56 of the tested isolates were susceptible to ampicillin, cephalothin, chloramphenicol, streptomycin, and tetracycline. For 45 isolates tested by agar dilution, the MIC of trimethoprim was 1 ug/rnl for 40 isolates, 0.3 ug/ml for three isolates, and 0.03 J.1g/ ml for two isolates. The MIC of sulfamethoxazole was 100J.1g/ml for 38 isolates, 30 ug/rnl for three isolates, and 10J.1g/ml for four isolates. When the drugs were combined, the MIC for 26 isolates was 0.1 ug of trimethoprim/rnl and 2 J.1g of sulfamethoxazole/ml; for 19 isolates the MIC was 0.03 J.1g of trimethoprim/ml and 0.6 J.1g of sulfamethoxazole/ ml. Blood cultures. Seventeen patients had bacteremia with colony counts ranging from < 10 to 4 X 107 colonies /ml. In four patients with colony counts of > 106/ml, bacteria were easily visible in the peripheral blood smears stained with Wright-Giemsa stain. Table 1 shows that 11 patients with colony counts of > 102/ml had higher incidences of hypotension and death than patients with lower colony counts and negative blood cultures. The patient who died without bacteremia had meningitis. Detection of endotoxemia. Ten patients studied in 1975, including five who were bactere-
Butler et al.
496
Table 2. Quantitative blood cultures, plasma limulus test results, and clinical features in 10 patients with Yersinia pestis infection. Plasma limulus test*
Quantitative blood culture (colonies/ml)
Acutephase specimen
Convalescentphase specimen
(21, F)
1 X 107
+
+
2
(28, F)
2 X 104
+
Femoral
3
(13, M)
Negative
4
(18, F)
