THE JOURNAL OF INFECTIOUS DISEASES· VOL. 132, NO.6· DECEMBER 1975 © 1975 by the University of Chicago. All rights reserved.
Comparison of Trimethoprim-Sulfamethoxazole and AmoxiciIIin in Therapy of Chloramphenicol-Resistant and ChloramphenicolSensitive Typhoid Fever From the Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland; Hospital Injectiologico CM. La Raw Hospital, Mexico City, Mexico; and lnstitucion Mexicana de Assistencia a la Ninez Hospital lnjantil, Mexico City, Mexico
Robert H. Gilman, Miguel Terminel, Myron M. Levine, Pablo Hernandez-Mendosa, Ernesto Calderone, Virginia Vasquez, Eloisa Martinez, Merrill J. Snyder, and Richard B. Hornick
The efficacy of orally administered trimethoprim-sulfamethoxazole was compared with that of oral amoxicillin in therapy of typhoid fever due to both epidemic chloramphenicol-resistant and endemic chloramphenicol-sensitive Salmonella ty phi. Both drug regimens were effective and of comparable value in treatment of chloramphenicol-resistant infections, as measured by duration of fever (124 hr and 115 hr, respectively) and duration of bacteremia (1.0 and 0.4 days, respectively). Trimethoprim-sulfamethoxazole therapy of infections due to chloramphenicol-sensitive S. typhi resulted in more rapid lysis of fever than did amoxicillin therapy. Trimethoprim and sulfamethoxazole were not synergistic in vitro against the chloramphenicol-resistant strain of S. typhi, and the role of sulfamethoxazole in treatment of such infections appears to be minimal. Oral administration of trimethoprim-sulfamethoxazole is effective therapy of chloramphenicol-resistant, and probably of arnpicillin-amoxicillin-resistant, typhoid fever.
Chloramphenicol has been the mainstay of therapy of typhoid fever for a quarter of a century. Its clinical efficacy, reliable gastrointestinal absorption, and relatively low cost make it suitable for use in typhoid-endemic, developing areas where economics and lack of facilities preclude routine hospitalization and iv medication for treatment of typhoid fever. Despite widespread, often indiscriminate use of chloramphenicol in areas of typhoid endemicity, resistance of Salmonella typhi to this antibiotic was sporadic and of little public health importance prior to 1972 [1-4J. In 1972, however, epidemic disease due to chloramphenicol-resistant (Cm") S. typhi appeared in Mexico [5-9J and then in Vietnam [10J. In both instances an R-factor was implicated that conferred resistance to chloramphenicol, sulfonamides, tetracycline, and streptomycin [4-7J. The coincidental appearance of widespread resistance of S. t yphi
to chloramphenicol in two far-flung areas of the world constituted a notable public health threat and emphasized the need for a reliable, alternative oral drug. Parenteral ampicillin [8, 11, 12] and oral amoxicillin [13], an ampicillin analogue, were found to be effective. However, the picture was complicated during the Mexican epidemic by sporadic isolation of S. typhi resistant to ampicillin as well as to chloramphenicol. Olarte [6J demonstrated that an additional episome, distinct from that conferring chloramphenicol resistance, mediated resistance to ampicillin; widespread resistance to ampicillin and amoxicillin, by dint of R-factor transfer, became a distinct possibility. Search for an oral antibiotic to replace both chloramphenicol and amoxicillin was deemed necessary. Trimethoprim-sulfamethoxazole (TMPSMZ) was chosen for evaluation of its success in treatment of disease caused by chloramphenicolsensitive S. typhi.
Received for publication February 26, 1975, and in revised form July 22, 1975. Please address requests for reprints to Dr. Robert H. Gilman, Division of Infectious Diseases, University of Maryland School of Medicine, 29 South Greene Street, Baltimore, Maryland 21201.
Patients and Methods
The study was conducted in two hospitals in Mexico City, Hospital Infectiologico C.M. La 630
Chloramphenicol-Resistant Typhoid Fever
Raza and Institucion Mexicana de Assistencia a la Ninez Hospital Infantil (IMAN), from May through July 1973. Patients included 20 children, aged six to 14 years, and 44 adults, aged 15-47 years, admitted with a clinical diagnosis of typhoid fever. Excluded were patients unable to take oral medication during the first 48 hr after admission, those with severe complications of typhoid fever, those with a history of allergy to penicillin or sulfonamides, those with disseminated intravascular coagulopathy, those who received concomitant steroid therapy, or patients in relapse. Also excluded were patients who had clinical signs of typhoid fever but from whom S. typhi was not recovered bacteriologically. Patients were randomly allocated to one of two groups and were treated for 10 days with TMPSMZ or with amoxicillin. Amoxicillin (6[D( -)y-amino-p-hydroxyphenylactamido] penicillanic acid; a-amino-p-hydroxybenzylpenicillin), an analogue of ampicillin with identical antibacterial spectrum, was administered to the control patients because high blood levels follow oral administration of this drug (twice those of ampicillin) [17, 18] and its efficacy has been proven in the treatment of chloramphenicol-resistant typhoid fever in Mexico [13]. TMP-SMZ tablets in fixed combination (80 mg of TMP plus 400 mg of SMZ per tablet) were administered in dosages of 8 mg/kg and 40 mg/kg, respectively, to the nearest half tablet per 24 hr in three divided doses. The maximal dosage was six tablets daily. Amoxicillin (100 mg/kg per 24 hr) was given in four divided doses up to a maximum of 6 g daily. Laboratory studies. Before initiation of therapy, blood, bone marrow, and stool were obtained from all patients for culture [19]; skin lesions suspected to be rose spots were also cultured [19, 20]. Samples of blood (2 ml) were inoculated into supplemented peptone broth (Vacutainer culture tubes; Becton-Dickinson, Rutherford, N.J.); marrow was cultured in supplemented peptone broth and in Ruiz-Castaneda bottles [21]. Rectal swabs were inoculated into selenite enrichment broth, which was subcultured onto both salmonella-shigella and MacConkey's agar. As a test of the efficacy of the antibiotics, additional blood cultures were obtained daily during the first five days of therapy and again two days after therapy ended. Rectal swabs were obtained
631
on days 3, 5, 9, and 12 or 14 after initiation of therapy. Suspect isolates were identified as S. typhi by standard methods [22]. Complete blood cell counts (including platelets and reticulocytes), blood urea nitrogen, serum creatinine, and liver function tests were performed at admission and on day 12 of hospitalization. A urinalysis and a chest radiograph were done at admission. Isolates of S. typhi were examined for susceptibility to chloramphenicol, ampicillin, tetracycline, streptomycin, and TMP-SMZ [23]. MICs to TMP, ampicillin, chloramphenicol, and SMZ were determined by twofold dilution techniques. A modification of the checkerboard technique of Bushby [24] was utilized to evaluate synergism between TMP and SMZ; in the assay used, 2 % lysed horse blood was added to Mueller-Hinton broth. Trough (O-min) and peak (120-min) concentrations of antibiotics in serum were determined on selected days after initiation of therapy in a number of patients in each therapeutic group. Clinical studies. Patients were observed in the hospital for two to four days after completion of antibiotic therapy. Oral temperatures were obtained every 4 hr throughout hospitalization. Total hours of fever was defined as the number of hours in which fever was sustained above 37.8 C. Single temperature spikes that occurred beyond 24 hr after the end of the febrile period were not added to the total hours of fever. For analysis of the data, typhoid fever was defined as an acute illness, with signs and symptoms compatible with typhoid, in addition to bacteriologic confirmation of the diagnosis by isolation of S. typhi from blood, bone marrow, rose spot, or stool. Results
In total, 64 patients whose illness met the criteria for typhoid fever were studied. The 34 patients in the group treated with TMP-SMZ were similar to the 30 treated with amoxicillin with respect to age, sex, duration of illness, prior antibiotic therapy, hematologic values, and liver function (table 1 ) . Two patients were unable to complete the 10 days of therapy and were not considered in the final analysis. One of these two patients received TMP-SMZ and exhibited severe thrombocytope-
Gilman et al.
632
Table 1. Clinical findings on admission of patients with typhoid fever who were treated with amoxicillin or trimethoprim-sulfamethoxazole (TMP-SMZ). Therapy (no. of patients) Amoxicillin (30) TMP-SMZ (34)
Mean duration of disease (days)
Mean hematocrit
t1.6 11.2
No. with positive cultures on admissiont
(% )
Mean WBC (X tOil/mm 3 ) *
Blood
Stool
42 41
6.3 6.8
12 15
15 14
* WBC = white
blood cell count. t Bacteriologic confirmation in the remaining cases was made by recovery of Salmonella typhi from bone marrow or rose spots [19l.
nia. The other patient received amoxicillin and developed nausea, vomiting, and diarrhea on the second day of therapy. These signs. continued unabated and were accompanied by increasing toxemia, necessitating a change to iv therapy with ampicillin and steroids on the fourth day. Blood cultures were positive for the first four days of therapy with amoxicillin and became negative 24 hr after iv administration of antibiotic. The concentration of amoxicillin in serum on the second day of therapy, 2 hr after oral administration of the drug, was 23 ug/rnl. Of the 62 patients who completed therapy, the group receiving amoxicillin included 18 individuals infected with the epidemic Cm R strain and 11 infected with chloramphenicolsensitive (Cm S ) endemic strains; the group treated with TMP-SMZ was composed of 23 patients with Cm R infection and 10 infected with Crn" strains.
Antibiotic sensitivities of isolated bacteria. The MICs of chloramphenicol for all Cm" isolates were >400 ug/rnl, whereas Cm" strains were inhibited by ~25 ug of chloramphenicol/ml (mean MIC, 6.6 ug/rnl). A concentration of TMP ~ 1 ug/ ml was inhibitory for both epidemic and endemic strains (i.e., strains with or without an R-factor), with no difference in susceptibility. There was, however, a marked difference in sensitivity to SMZ of the endemic R-factor-deficient (R -) strains compared with the epidemic R-factor-containing (R +) isolates. The endemic Cms (R -) isolates were uniformly inhibited by ~62 ug of SMZ/ml. In contrast, the epidemic Cm R (R +) isolates were all resistant to > 62 !Ag of SMZ, tetracycline, or streptomycin/ml. Five randomly selected epidemic (R +) isolates were resistant to > 250 !Ag of SMZ/ml. Synergism of the TMP-SMZ combination has
been defined as at least a fourfold reduction of the MIC to one of the two drugs and at least a twofold reduction of the MIC to the other. Tube dilutions utilizing the combination of TMP-SMZ in a ratio of 1: 20 revealed evidence of synergism in the endemic Crn", sulfonamide-sensitive strains, whereas no synergism was detected with epidemic Cm R , sulfonamide-resistant isolates. These findings were confirmed by use of the checkerboard technique in assay of eight endemic Cm", sulfonamide-sensitive strains and of six epidemic Cm R , sulfonamide-resistant isolates. A fourfold decrease in the MICs of both TMP and SMZ was found for eight sensitive strains but not for any of the six resistant strains. All 64 strains of S. typhi isolated in initial cultures were sensitive to 1.6 ug of ampicillirr/rnl (mean MIC, 0.8 ug/ml) , An ampicillin-resistant strain (MIC, 50 ug/rnl ) was recovered from the stool culture of one patient in the amoxicillin-treated group after three days of therapy, at which time sensitive organisms had already been eliminated from blood cultures. Response of patients. The patients from whom Cm" (R -) endemic strains were isolated were clinically similar to patients infected with the Cm R (R +) epidemic strain (table 2). A total of 62 patients completed 10 days of oral therapy. The mean number of fever hours and mean number of days of positive blood culture after initiation of therapy were used as objective measurements for comparison of the efficacy of the two antibiotic therapies. Patients in the amoxicillin-treated group remained febrile longer than patients in the TMPSMZ-treated group (table 3); the difference in mean number of fever hours (128 vs. 105) was not statistically significant, however (P == 0.08). Within each group the clinical response of patients infected with the epidemic (R +) strain was compared with that of patients infected with the
Chloramphenicol-Resistant Typhoid Fever
633
Table 2. Clinical characteristics of patients infected with epidemic chloramphenicol-resistant and endemic chloramphenicol-sensitive strains of Salmonella typhi. Mean No. (%) with duration Mean Mean of disease hematocrit Palpable Palpable WBC (days) (% ) spleen (X 103/mm3) * liver
Strain (no. of patients) Epidemic (R +) resistant (42) Endemic (R -) sensitive (22)
* WBC =
11.4 11.6
18 (43) 8 (36)
6.4 6.6
41 41
No. (%) with positive cultures on admission
10 (22) 3 (13)
Blood
Stool
20 (48) 7 (32)
22 (52) 7 (32)
white blood cell count.
(R -) endemic strains. Among patients receiving amoxicillin, the mean febrile period after initiation of therapy was quite similar for the two subpopulations. However, among the patients who received TMP-SMZ, the mean number of fever hours was greater in those infected with the epidemic strains (115) than in those harboring the endemic strains (83); the difference was suggestive, but not significant (P = 0.09). Patients infected with endemic strains and given TMPSMZ had a significantly shorter febrile period (83 hr) than those who received amoxicillin (136 hr) (P = 0.005). Analysis of daily blood culture results offered another objective means of comparison (table 3). Patients in the amoxicillin-treated group had positive blood cultures for a mean of 1.1 days after initiation of therapy, while those receiving TMP-SMZ had positive blood cultures for a mean of 0.4 days after therapy was begun. The bacteriologic response of patients infected
with either the multiply sensitive endemic strains or the multiply resistant epidemic strain was comparable within each therapeutic group (table 3). Patients infected with either the epidemic strain or endemic strains and treated with TMPSMZ had somewhat more rapid clearance of bacteremia than corresponding patients treated with amoxicillin, although the differences were significant (P = 0.05) only when the data from all patients were combined (table 3). The rates of clearance of S. typhi from stool cultures were comparable for the two groups. Prior to commencement of therapy, approximately half of the patients in each group had positive stool cultures. After nine days of therapy, none of 29 patients in the amoxicillin-treated group and two of 29 in the TMP-SMZ-treated group had positive stool cultures. After 12 days of therapy, two of 28 in the amoxicillin-treated group and none of 32 in the TMP-SMZ-treated group had positive cultures. The frequency of relapse in the two therapeutic
Table 3. Comparison of mean number of fever hours and duration of bacteremia in patients with typhoid fever who were treated with amoxicillin or with trimethoprim-sulfamethoxazole (TMP-SMZ). Antibiotic (no. treated)
Group
Mean no. of fever hours during therapy
= 0.08t
13
16
1.1 0.4
P
= 0.57
7 13
1.0 0.4
3 6
0.3 1.2
Amoxicillin (29) TMP·SMZ (33)
Patients infected with the epidemic (R +) strain
Amoxicillin (18)t TMP-SMZ (23)
115
TMP-SMZ (10) Amoxicillin (11) t
} P 83 P 136
105
124
Mean duration of bacteremia during therapy
P
All patients with typhoid fever
Patients infected with the endemic (R -) strains
128
No. with positive blood cultures*
P =0.05
P
= 0.20
P
= 0.30
= 0.09 0005
=.
* A total of 29 blood cultures are included (one patient with positive blood culture on admission was excluded from analysis because of failure to complete 10 days of therapy). Three patients had positive blood cultures after therapy started but had negative cultures initially. t Student's t-test. +The mean duration of fever and that of bacteremia were not significantly different in patients who had endemic strains and were treated with amoxicillin from these values in patients who had the epidemic strain and were treated with amoxicillin (P 0.49 and 0.81, respectively).
=
634
Gilman et al.
groups could not be calculated because the majority of patients could not be followed after discharge. Patients were queried with respect to antibiotic ingestion before admission, since across-thecounter sales and self-therapy promote promiscuous antibiotic usage in this population. When patients who had received an appropriate antibiotic prior to admission were compared with those who took inappropriate or no antibiotics, no significant differences were found in frequency of positive blood or stool cultures on admission or in response to therapy with amoxicillin or TMPSMZ. Concentrations of antibiotics in serum. Concentrations of amoxicillin, TMP, and SMZ in serum 2 hr after oral administration of drug on days 0, 2, 5, and 10 were comparable to those reported by other investigators [26, 27] (table 4). Amoxicillin and TMP levels surpassed the MICs for all strains, both with and without R-factors. Levels of SMZ were not sufficient to surpass the MICs for epidemic Cm R , sulfonamide-resistant strains. Adverse reactions to drugs. One patient among the 34 who began TMP-SMZ therapy developed severe thrombocytopenia on the seventh day of therapy, at which time his clinical response was excellent. TMP-SMZ was discontinued and his platelet count rose; he then had a recurrence of typhoid. After seven days without antibiotics, during which time his platelet count rose, this patient was given ampicillin, which led to recovery. Six of the 30 patients who received amoxicillin developed an erythematous rash. Therapy was continued in all instances without incident.
Discussion
The results indicate that TMP-SMZ compares favorably with amoxicillin as a reasonable alternative for therapy of Cm R typhoid fever when oral medication can be tolerated. Although ampicillin-resistant strains did not become an important public health problem subsequent to their initial identification [6] and although we did not encounter any such isolates prior to therapy in this series, we believe TMP-SMZ to be the rational therapy for ampicillin-resistant typhoid fever until proven otherwise. The role of SMZ in the TMP-SMZ combination in treatment of Mexican Cm R (R +) typhoid is unclear; in vitro levels of resistance are very high, and we, like Anderson [28], could demonstrate no synergism when SMZ was added to TMP. The lack of synergism against the R + strain was apparently evident in vivo; the mean duration of fever in Cm R , sulfonamide-resistant infections treated with TMP-SMZ was 115 hr, as compared with 83 hr in the Crn", sulfonamide-sensitive cases. Thus SMZ had a marked, albeit not significant (P == 0.09), effect on the antibiotic-sensitive strains as compared with the multiply resistant strains; this observation suggests synergism in the latter type of strain. In contrast, the duration of fever was quite similar for both Cm R and Cm" infections treated with amoxicillin. Clinical trials comparing TMP alone to TMP-SMZ are needed to establish the role of SMZ in therapy of Cm" and Cm R typhoid fever. SMZ, rather than TMP, is presumably responsible for most of the nonhematologic toxicity that follows administration of this combination [29]. In our study, as well as
Table 4.
Levels of antibiotics in sera of patients with typhoid fever who were treated with trimethoprim (TMP), sulfamethoxazole (SMZ). or amoxicillin. Day
Antibiotic
0
2
5
10
NO*
14.8 ± 4.93 (/I 28)
=
15.5 ::±: 5.62 (/I 24)
NO
TMP
1.0 ± 0.69 (/I 17)
=
2.8 ± 1.5 (/I 28)
=
2.9 ± 0.78 (n 28)
=
2.7 ::±: 0.78 (II 19)
SMZ
13.2 ± 11.3 (/I 17)
78.2 ± 9.2 in 28)
95.9 ± 29.6 (n 28)
113.2 ± 28.8 (n 19)
Amoxicillin
=
=
=
=
=
=
NOTE. Values given are mean concentrations (± SD) of antibiotic determined in serum 2 hr after administration of the drug and expressed as ug./ml. * NO not determined.
=
Chloramphenicol-Resistant Typhoid Fever
in that of Snyder et a1. [27], SMZ accumulated over time in the blood of patients with typhoid fever treated with TMP-SMZ. The one drawback to use of TMP-SMZ that we encountered was the severe thrombocytopenia in one of 34 patients who received TMP-SMZ. This is a known complication of TMP-SMZ therapy and is apparently due to the effects of TMP on marrow precursor cells [30]. Concomitant therapy with folinic acid has been suggested as a means by which the hematologic effects in humans may be prevented without decreasing antibacterial activity [30]. Chloramphenicol remains the drug of choice for typhoid fever where resistant strains are not prevalent. Either TMP-SMZ or amoxicillin appears to be a rational and effective choice for therapy of typhoid fever where Cm R strains are common and in travelers infected in such areas. TMP-SMZ is also suggested as an alternative for therapy of infection with ampicillin-resistant Cm R strains. References 1. Colguhoun, J., Weetch, R. S. Resistance to chloramphenicol developing during treatment of typhoid fever. Lancet 2:621-623, 1950. 2. MUfti, R. B., Rajyalakshmi, K, Bhaskaran, C. S. Resistance of Salmonella typhi to chloramphenicol. Part I. A preliminary report. J. Clin. Pathol. 15: 544-551, 1962. 3. Njoku-Obi, A. N., Njoku-Obi, J. C. Resistance of Sa/monel/a typhosa to chloramphenicol. J. Bacteriol. 90:552-553, 1965. 4. Anderson, E. S., Smith, H. R. Chloramphenicol resistance in the typhoid bacillus. Br. Med. J. 3: 329-331, 1972. 5. Gangarosa, E. J., Bennett, J. V., Wyatt, c, Pierce, P. E., Olarte, J., Mendoza, P. H., Vasquez. V., Bessudo, D. M. An epidemic-associated episome? J. Infect. Dis. 126:215-218, 1972. 6. Olarte, J., Galindo, E. Salmonella typhi resistant to chloramphenicol, ampicillin and other antimicrobial agents: strains isolated during an extensive typhoid fever epidemic in Mexico. Antimicrob. Agents Chemother. 4: 596-601, 1973. 7. Bessudo, D. M., Olarte, J., Mendoza-Hernandez, P., Galindo, E., Carrillo, J., Gutierrez, Trujillo, G., Kurnate, J. Aislamiento de S. typhi resistente a altas concentraciones de chloranfenicol. Boletin de la Oficinia Sanitaria Panamericana 74: 1-6, 1973. . 8. Overturf, G., Marton, K I., Mathies, A. W., Jr. Antibiotic resistance in typhoid fever. N. Engl. J. Med. 289:463-465, 1973.
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9. Gonzalez-Cortes, A., Bessudo, D., Sanchez-Leyva, R., Fragoso, R., Hinojosa, M., Becerril, P. Waterborne transmission of cloramphenicol-resistant Salmonella typhi in Mexico. Lancet 2:605-607, 1973. 10. Butler, T., Linh, N. N., Ai nold, K, Pollack, M. Chloramphenicol-resistant typhoid fever in Vietnam associated with R factor. Lancet 2: 983-985, 1973. 11. Calderon, E., Gilman, R. H., Snyder, M. J., Vasquez, V., Gomez Barreto, D., Legorreta, J., Rodriguez, R., Martinez, E., Hornick, R. B., Woodward, T. E. A controlled clinical trial of chloramphenicol in chloramphenicol-resistant typhoid fever. Rev. Lat. Am. Microbiol. 16:131-136, 1974. 12. Center for Disease Control. Typhoid fever contracted in Mexico. Morbidity and Mortality Weekly Rep. 21:193-194,1972. 13. Calderon, E. Amoxicillin in the treatment of typhoid fever due to chloramphenicol-resistant Salmonella typhi. J. Infect. Dis. 129(Suppl.) :S219-S222, 1974. 14. Kamat, S. A. Evaluation of therapeutic efficacy of trimethoprim-sulfamethoxazole and chloramphenicol in enteric fever. Br. Med. J. 3: 320-322, 1970. 15. Stamps, T. J., Wicks, A. C. B. Trirnethoprim-sulfamethoxazole (Bactrim) in the treatment of typhoid fever. S. Afr. Med. J. 46: 652-655, 1972. 16. Cardoso, N. Double-blind trial with chloramphenicol and the combination trimethoprim-sulfamethoxazole in typhoid. S. Afr. Med. J. 46: 1286-1287, 1972. 17. Gordon, R. c., Regamey, c., Kirby, W. M. Comparative clinical pharmacology of amoxicillin and ampicillin administered orally. Antimicrob. Agents Chemother. I: 504-507, 1972. 18. Neu, H. c, Winshell, E. B. Pharmacological studies of 6 [D( - )-a-amino-p-hydroxyphenylacetamido] penicillanic acid in humans. Antimicrob. Agents Chemother. 1970: 423-426, 1971. 19. Gilman, R. H., Terrninel, M., Levine, M. M., Hernandez-Mendoza, P., Hornick, R. B. Relative efficacy of blood, urine, rectal swab, bone marrow, and rose-spot cultures for recovery of Salmonella typhi in typhoid fever. Lancet 1:1211-1213,1975. 20. Terminel Valenzuela, N., Mendoza Hernandez, P., Bahena Albarran, I. Tecnica del cultivo de la biopsia de roseola para el diagnostico de la fiebre tifoidea. Rev. Lat. Am. Microbiol. 15:69-70, 1973. 21. Castaneda, M. R. Practical method for routine blood cultures in brucellosis. Proc. Soc. Exp. BioI. Med. 64: I 14-115, 1947. 22. Edwards, P. R., Ewing, W. H. [ed.]. Identification of Enterobacteriaceae. Burgess, Minneapolis, 1962, p. 168-173. 23. Bauer, A. W., Kirby, W. M. M., Sherris, J. c, Turck, M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496, 1966.
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24. Bushby, S. R. Sensitivity testing with trimethoprirnsulfamethoxazole. Med. J. Aust. 1(Suppl.): 10-18, 1973. 25. Bach, M. c, Finland, M., Gold, 0., Wilcox, C. Susceptibility of recently isolated pathogenic bacteria to trimethoprim and sulfamethoxazole separately and combined. J. Infect. Dis. 128(Suppl.): S508-S533, 1973. 26. Neu, H. C. Antimicrobial activity and human pharmacology of amoxicillin. J. Infect. Dis. 129 (Suppl) :SI23-8133, 1974. 27. Snyder, M. J., Perroni, J., Gonzalez, 0., Palomino,
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c., Gonzalez, c., Music, S., DuPont, H. L., Hornick, R. B., Woodward, T. E. Trimethoprim-sulfamethoxazole in the treatment of typhoid and paratyphoid fevers. J. Infect. Dis. 128(Suppl): S734-8737, 1973. 28. Anderson, E. S. Chloramphenicol-resistant Salmonella typhi. Lancet 2: 1494-1495, 1973. 29. Garrod, L. P. Trimethoprim: its possible place in antibacterial therapy. Drugs 1: 3-6, 1971. 30. Kahn, S. B., Fein, S. A., Brodsky, I. Effects of trimethoprim on folate metabolism in man. Clin. Pharmacal. Ther. 9: 550-560, 1968.
Comparison of Trimethoprim-Sulfamethoxazole and AmoxiciIIin in Therapy of Chloramphenicol-Resistant and ChloramphenicolSensitive Typhoid Fever From the Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland; Hospital Injectiologico CM. La Raw Hospital, Mexico City, Mexico; and lnstitucion Mexicana de Assistencia a la Ninez Hospital lnjantil, Mexico City, Mexico
Robert H. Gilman, Miguel Terminel, Myron M. Levine, Pablo Hernandez-Mendosa, Ernesto Calderone, Virginia Vasquez, Eloisa Martinez, Merrill J. Snyder, and Richard B. Hornick
The efficacy of orally administered trimethoprim-sulfamethoxazole was compared with that of oral amoxicillin in therapy of typhoid fever due to both epidemic chloramphenicol-resistant and endemic chloramphenicol-sensitive Salmonella ty phi. Both drug regimens were effective and of comparable value in treatment of chloramphenicol-resistant infections, as measured by duration of fever (124 hr and 115 hr, respectively) and duration of bacteremia (1.0 and 0.4 days, respectively). Trimethoprim-sulfamethoxazole therapy of infections due to chloramphenicol-sensitive S. typhi resulted in more rapid lysis of fever than did amoxicillin therapy. Trimethoprim and sulfamethoxazole were not synergistic in vitro against the chloramphenicol-resistant strain of S. typhi, and the role of sulfamethoxazole in treatment of such infections appears to be minimal. Oral administration of trimethoprim-sulfamethoxazole is effective therapy of chloramphenicol-resistant, and probably of arnpicillin-amoxicillin-resistant, typhoid fever.
Chloramphenicol has been the mainstay of therapy of typhoid fever for a quarter of a century. Its clinical efficacy, reliable gastrointestinal absorption, and relatively low cost make it suitable for use in typhoid-endemic, developing areas where economics and lack of facilities preclude routine hospitalization and iv medication for treatment of typhoid fever. Despite widespread, often indiscriminate use of chloramphenicol in areas of typhoid endemicity, resistance of Salmonella typhi to this antibiotic was sporadic and of little public health importance prior to 1972 [1-4J. In 1972, however, epidemic disease due to chloramphenicol-resistant (Cm") S. typhi appeared in Mexico [5-9J and then in Vietnam [10J. In both instances an R-factor was implicated that conferred resistance to chloramphenicol, sulfonamides, tetracycline, and streptomycin [4-7J. The coincidental appearance of widespread resistance of S. t yphi
to chloramphenicol in two far-flung areas of the world constituted a notable public health threat and emphasized the need for a reliable, alternative oral drug. Parenteral ampicillin [8, 11, 12] and oral amoxicillin [13], an ampicillin analogue, were found to be effective. However, the picture was complicated during the Mexican epidemic by sporadic isolation of S. typhi resistant to ampicillin as well as to chloramphenicol. Olarte [6J demonstrated that an additional episome, distinct from that conferring chloramphenicol resistance, mediated resistance to ampicillin; widespread resistance to ampicillin and amoxicillin, by dint of R-factor transfer, became a distinct possibility. Search for an oral antibiotic to replace both chloramphenicol and amoxicillin was deemed necessary. Trimethoprim-sulfamethoxazole (TMPSMZ) was chosen for evaluation of its success in treatment of disease caused by chloramphenicolsensitive S. typhi.
Received for publication February 26, 1975, and in revised form July 22, 1975. Please address requests for reprints to Dr. Robert H. Gilman, Division of Infectious Diseases, University of Maryland School of Medicine, 29 South Greene Street, Baltimore, Maryland 21201.
Patients and Methods
The study was conducted in two hospitals in Mexico City, Hospital Infectiologico C.M. La 630
Chloramphenicol-Resistant Typhoid Fever
Raza and Institucion Mexicana de Assistencia a la Ninez Hospital Infantil (IMAN), from May through July 1973. Patients included 20 children, aged six to 14 years, and 44 adults, aged 15-47 years, admitted with a clinical diagnosis of typhoid fever. Excluded were patients unable to take oral medication during the first 48 hr after admission, those with severe complications of typhoid fever, those with a history of allergy to penicillin or sulfonamides, those with disseminated intravascular coagulopathy, those who received concomitant steroid therapy, or patients in relapse. Also excluded were patients who had clinical signs of typhoid fever but from whom S. typhi was not recovered bacteriologically. Patients were randomly allocated to one of two groups and were treated for 10 days with TMPSMZ or with amoxicillin. Amoxicillin (6[D( -)y-amino-p-hydroxyphenylactamido] penicillanic acid; a-amino-p-hydroxybenzylpenicillin), an analogue of ampicillin with identical antibacterial spectrum, was administered to the control patients because high blood levels follow oral administration of this drug (twice those of ampicillin) [17, 18] and its efficacy has been proven in the treatment of chloramphenicol-resistant typhoid fever in Mexico [13]. TMP-SMZ tablets in fixed combination (80 mg of TMP plus 400 mg of SMZ per tablet) were administered in dosages of 8 mg/kg and 40 mg/kg, respectively, to the nearest half tablet per 24 hr in three divided doses. The maximal dosage was six tablets daily. Amoxicillin (100 mg/kg per 24 hr) was given in four divided doses up to a maximum of 6 g daily. Laboratory studies. Before initiation of therapy, blood, bone marrow, and stool were obtained from all patients for culture [19]; skin lesions suspected to be rose spots were also cultured [19, 20]. Samples of blood (2 ml) were inoculated into supplemented peptone broth (Vacutainer culture tubes; Becton-Dickinson, Rutherford, N.J.); marrow was cultured in supplemented peptone broth and in Ruiz-Castaneda bottles [21]. Rectal swabs were inoculated into selenite enrichment broth, which was subcultured onto both salmonella-shigella and MacConkey's agar. As a test of the efficacy of the antibiotics, additional blood cultures were obtained daily during the first five days of therapy and again two days after therapy ended. Rectal swabs were obtained
631
on days 3, 5, 9, and 12 or 14 after initiation of therapy. Suspect isolates were identified as S. typhi by standard methods [22]. Complete blood cell counts (including platelets and reticulocytes), blood urea nitrogen, serum creatinine, and liver function tests were performed at admission and on day 12 of hospitalization. A urinalysis and a chest radiograph were done at admission. Isolates of S. typhi were examined for susceptibility to chloramphenicol, ampicillin, tetracycline, streptomycin, and TMP-SMZ [23]. MICs to TMP, ampicillin, chloramphenicol, and SMZ were determined by twofold dilution techniques. A modification of the checkerboard technique of Bushby [24] was utilized to evaluate synergism between TMP and SMZ; in the assay used, 2 % lysed horse blood was added to Mueller-Hinton broth. Trough (O-min) and peak (120-min) concentrations of antibiotics in serum were determined on selected days after initiation of therapy in a number of patients in each therapeutic group. Clinical studies. Patients were observed in the hospital for two to four days after completion of antibiotic therapy. Oral temperatures were obtained every 4 hr throughout hospitalization. Total hours of fever was defined as the number of hours in which fever was sustained above 37.8 C. Single temperature spikes that occurred beyond 24 hr after the end of the febrile period were not added to the total hours of fever. For analysis of the data, typhoid fever was defined as an acute illness, with signs and symptoms compatible with typhoid, in addition to bacteriologic confirmation of the diagnosis by isolation of S. typhi from blood, bone marrow, rose spot, or stool. Results
In total, 64 patients whose illness met the criteria for typhoid fever were studied. The 34 patients in the group treated with TMP-SMZ were similar to the 30 treated with amoxicillin with respect to age, sex, duration of illness, prior antibiotic therapy, hematologic values, and liver function (table 1 ) . Two patients were unable to complete the 10 days of therapy and were not considered in the final analysis. One of these two patients received TMP-SMZ and exhibited severe thrombocytope-
Gilman et al.
632
Table 1. Clinical findings on admission of patients with typhoid fever who were treated with amoxicillin or trimethoprim-sulfamethoxazole (TMP-SMZ). Therapy (no. of patients) Amoxicillin (30) TMP-SMZ (34)
Mean duration of disease (days)
Mean hematocrit
t1.6 11.2
No. with positive cultures on admissiont
(% )
Mean WBC (X tOil/mm 3 ) *
Blood
Stool
42 41
6.3 6.8
12 15
15 14
* WBC = white
blood cell count. t Bacteriologic confirmation in the remaining cases was made by recovery of Salmonella typhi from bone marrow or rose spots [19l.
nia. The other patient received amoxicillin and developed nausea, vomiting, and diarrhea on the second day of therapy. These signs. continued unabated and were accompanied by increasing toxemia, necessitating a change to iv therapy with ampicillin and steroids on the fourth day. Blood cultures were positive for the first four days of therapy with amoxicillin and became negative 24 hr after iv administration of antibiotic. The concentration of amoxicillin in serum on the second day of therapy, 2 hr after oral administration of the drug, was 23 ug/rnl. Of the 62 patients who completed therapy, the group receiving amoxicillin included 18 individuals infected with the epidemic Cm R strain and 11 infected with chloramphenicolsensitive (Cm S ) endemic strains; the group treated with TMP-SMZ was composed of 23 patients with Cm R infection and 10 infected with Crn" strains.
Antibiotic sensitivities of isolated bacteria. The MICs of chloramphenicol for all Cm" isolates were >400 ug/rnl, whereas Cm" strains were inhibited by ~25 ug of chloramphenicol/ml (mean MIC, 6.6 ug/rnl). A concentration of TMP ~ 1 ug/ ml was inhibitory for both epidemic and endemic strains (i.e., strains with or without an R-factor), with no difference in susceptibility. There was, however, a marked difference in sensitivity to SMZ of the endemic R-factor-deficient (R -) strains compared with the epidemic R-factor-containing (R +) isolates. The endemic Cms (R -) isolates were uniformly inhibited by ~62 ug of SMZ/ml. In contrast, the epidemic Cm R (R +) isolates were all resistant to > 62 !Ag of SMZ, tetracycline, or streptomycin/ml. Five randomly selected epidemic (R +) isolates were resistant to > 250 !Ag of SMZ/ml. Synergism of the TMP-SMZ combination has
been defined as at least a fourfold reduction of the MIC to one of the two drugs and at least a twofold reduction of the MIC to the other. Tube dilutions utilizing the combination of TMP-SMZ in a ratio of 1: 20 revealed evidence of synergism in the endemic Crn", sulfonamide-sensitive strains, whereas no synergism was detected with epidemic Cm R , sulfonamide-resistant isolates. These findings were confirmed by use of the checkerboard technique in assay of eight endemic Cm", sulfonamide-sensitive strains and of six epidemic Cm R , sulfonamide-resistant isolates. A fourfold decrease in the MICs of both TMP and SMZ was found for eight sensitive strains but not for any of the six resistant strains. All 64 strains of S. typhi isolated in initial cultures were sensitive to 1.6 ug of ampicillirr/rnl (mean MIC, 0.8 ug/ml) , An ampicillin-resistant strain (MIC, 50 ug/rnl ) was recovered from the stool culture of one patient in the amoxicillin-treated group after three days of therapy, at which time sensitive organisms had already been eliminated from blood cultures. Response of patients. The patients from whom Cm" (R -) endemic strains were isolated were clinically similar to patients infected with the Cm R (R +) epidemic strain (table 2). A total of 62 patients completed 10 days of oral therapy. The mean number of fever hours and mean number of days of positive blood culture after initiation of therapy were used as objective measurements for comparison of the efficacy of the two antibiotic therapies. Patients in the amoxicillin-treated group remained febrile longer than patients in the TMPSMZ-treated group (table 3); the difference in mean number of fever hours (128 vs. 105) was not statistically significant, however (P == 0.08). Within each group the clinical response of patients infected with the epidemic (R +) strain was compared with that of patients infected with the
Chloramphenicol-Resistant Typhoid Fever
633
Table 2. Clinical characteristics of patients infected with epidemic chloramphenicol-resistant and endemic chloramphenicol-sensitive strains of Salmonella typhi. Mean No. (%) with duration Mean Mean of disease hematocrit Palpable Palpable WBC (days) (% ) spleen (X 103/mm3) * liver
Strain (no. of patients) Epidemic (R +) resistant (42) Endemic (R -) sensitive (22)
* WBC =
11.4 11.6
18 (43) 8 (36)
6.4 6.6
41 41
No. (%) with positive cultures on admission
10 (22) 3 (13)
Blood
Stool
20 (48) 7 (32)
22 (52) 7 (32)
white blood cell count.
(R -) endemic strains. Among patients receiving amoxicillin, the mean febrile period after initiation of therapy was quite similar for the two subpopulations. However, among the patients who received TMP-SMZ, the mean number of fever hours was greater in those infected with the epidemic strains (115) than in those harboring the endemic strains (83); the difference was suggestive, but not significant (P = 0.09). Patients infected with endemic strains and given TMPSMZ had a significantly shorter febrile period (83 hr) than those who received amoxicillin (136 hr) (P = 0.005). Analysis of daily blood culture results offered another objective means of comparison (table 3). Patients in the amoxicillin-treated group had positive blood cultures for a mean of 1.1 days after initiation of therapy, while those receiving TMP-SMZ had positive blood cultures for a mean of 0.4 days after therapy was begun. The bacteriologic response of patients infected
with either the multiply sensitive endemic strains or the multiply resistant epidemic strain was comparable within each therapeutic group (table 3). Patients infected with either the epidemic strain or endemic strains and treated with TMPSMZ had somewhat more rapid clearance of bacteremia than corresponding patients treated with amoxicillin, although the differences were significant (P = 0.05) only when the data from all patients were combined (table 3). The rates of clearance of S. typhi from stool cultures were comparable for the two groups. Prior to commencement of therapy, approximately half of the patients in each group had positive stool cultures. After nine days of therapy, none of 29 patients in the amoxicillin-treated group and two of 29 in the TMP-SMZ-treated group had positive stool cultures. After 12 days of therapy, two of 28 in the amoxicillin-treated group and none of 32 in the TMP-SMZ-treated group had positive cultures. The frequency of relapse in the two therapeutic
Table 3. Comparison of mean number of fever hours and duration of bacteremia in patients with typhoid fever who were treated with amoxicillin or with trimethoprim-sulfamethoxazole (TMP-SMZ). Antibiotic (no. treated)
Group
Mean no. of fever hours during therapy
= 0.08t
13
16
1.1 0.4
P
= 0.57
7 13
1.0 0.4
3 6
0.3 1.2
Amoxicillin (29) TMP·SMZ (33)
Patients infected with the epidemic (R +) strain
Amoxicillin (18)t TMP-SMZ (23)
115
TMP-SMZ (10) Amoxicillin (11) t
} P 83 P 136
105
124
Mean duration of bacteremia during therapy
P
All patients with typhoid fever
Patients infected with the endemic (R -) strains
128
No. with positive blood cultures*
P =0.05
P
= 0.20
P
= 0.30
= 0.09 0005
=.
* A total of 29 blood cultures are included (one patient with positive blood culture on admission was excluded from analysis because of failure to complete 10 days of therapy). Three patients had positive blood cultures after therapy started but had negative cultures initially. t Student's t-test. +The mean duration of fever and that of bacteremia were not significantly different in patients who had endemic strains and were treated with amoxicillin from these values in patients who had the epidemic strain and were treated with amoxicillin (P 0.49 and 0.81, respectively).
=
634
Gilman et al.
groups could not be calculated because the majority of patients could not be followed after discharge. Patients were queried with respect to antibiotic ingestion before admission, since across-thecounter sales and self-therapy promote promiscuous antibiotic usage in this population. When patients who had received an appropriate antibiotic prior to admission were compared with those who took inappropriate or no antibiotics, no significant differences were found in frequency of positive blood or stool cultures on admission or in response to therapy with amoxicillin or TMPSMZ. Concentrations of antibiotics in serum. Concentrations of amoxicillin, TMP, and SMZ in serum 2 hr after oral administration of drug on days 0, 2, 5, and 10 were comparable to those reported by other investigators [26, 27] (table 4). Amoxicillin and TMP levels surpassed the MICs for all strains, both with and without R-factors. Levels of SMZ were not sufficient to surpass the MICs for epidemic Cm R , sulfonamide-resistant strains. Adverse reactions to drugs. One patient among the 34 who began TMP-SMZ therapy developed severe thrombocytopenia on the seventh day of therapy, at which time his clinical response was excellent. TMP-SMZ was discontinued and his platelet count rose; he then had a recurrence of typhoid. After seven days without antibiotics, during which time his platelet count rose, this patient was given ampicillin, which led to recovery. Six of the 30 patients who received amoxicillin developed an erythematous rash. Therapy was continued in all instances without incident.
Discussion
The results indicate that TMP-SMZ compares favorably with amoxicillin as a reasonable alternative for therapy of Cm R typhoid fever when oral medication can be tolerated. Although ampicillin-resistant strains did not become an important public health problem subsequent to their initial identification [6] and although we did not encounter any such isolates prior to therapy in this series, we believe TMP-SMZ to be the rational therapy for ampicillin-resistant typhoid fever until proven otherwise. The role of SMZ in the TMP-SMZ combination in treatment of Mexican Cm R (R +) typhoid is unclear; in vitro levels of resistance are very high, and we, like Anderson [28], could demonstrate no synergism when SMZ was added to TMP. The lack of synergism against the R + strain was apparently evident in vivo; the mean duration of fever in Cm R , sulfonamide-resistant infections treated with TMP-SMZ was 115 hr, as compared with 83 hr in the Crn", sulfonamide-sensitive cases. Thus SMZ had a marked, albeit not significant (P == 0.09), effect on the antibiotic-sensitive strains as compared with the multiply resistant strains; this observation suggests synergism in the latter type of strain. In contrast, the duration of fever was quite similar for both Cm R and Cm" infections treated with amoxicillin. Clinical trials comparing TMP alone to TMP-SMZ are needed to establish the role of SMZ in therapy of Cm" and Cm R typhoid fever. SMZ, rather than TMP, is presumably responsible for most of the nonhematologic toxicity that follows administration of this combination [29]. In our study, as well as
Table 4.
Levels of antibiotics in sera of patients with typhoid fever who were treated with trimethoprim (TMP), sulfamethoxazole (SMZ). or amoxicillin. Day
Antibiotic
0
2
5
10
NO*
14.8 ± 4.93 (/I 28)
=
15.5 ::±: 5.62 (/I 24)
NO
TMP
1.0 ± 0.69 (/I 17)
=
2.8 ± 1.5 (/I 28)
=
2.9 ± 0.78 (n 28)
=
2.7 ::±: 0.78 (II 19)
SMZ
13.2 ± 11.3 (/I 17)
78.2 ± 9.2 in 28)
95.9 ± 29.6 (n 28)
113.2 ± 28.8 (n 19)
Amoxicillin
=
=
=
=
=
=
NOTE. Values given are mean concentrations (± SD) of antibiotic determined in serum 2 hr after administration of the drug and expressed as ug./ml. * NO not determined.
=
Chloramphenicol-Resistant Typhoid Fever
in that of Snyder et a1. [27], SMZ accumulated over time in the blood of patients with typhoid fever treated with TMP-SMZ. The one drawback to use of TMP-SMZ that we encountered was the severe thrombocytopenia in one of 34 patients who received TMP-SMZ. This is a known complication of TMP-SMZ therapy and is apparently due to the effects of TMP on marrow precursor cells [30]. Concomitant therapy with folinic acid has been suggested as a means by which the hematologic effects in humans may be prevented without decreasing antibacterial activity [30]. Chloramphenicol remains the drug of choice for typhoid fever where resistant strains are not prevalent. Either TMP-SMZ or amoxicillin appears to be a rational and effective choice for therapy of typhoid fever where Cm R strains are common and in travelers infected in such areas. TMP-SMZ is also suggested as an alternative for therapy of infection with ampicillin-resistant Cm R strains. References 1. Colguhoun, J., Weetch, R. S. Resistance to chloramphenicol developing during treatment of typhoid fever. Lancet 2:621-623, 1950. 2. MUfti, R. B., Rajyalakshmi, K, Bhaskaran, C. S. Resistance of Salmonella typhi to chloramphenicol. Part I. A preliminary report. J. Clin. Pathol. 15: 544-551, 1962. 3. Njoku-Obi, A. N., Njoku-Obi, J. C. Resistance of Sa/monel/a typhosa to chloramphenicol. J. Bacteriol. 90:552-553, 1965. 4. Anderson, E. S., Smith, H. R. Chloramphenicol resistance in the typhoid bacillus. Br. Med. J. 3: 329-331, 1972. 5. Gangarosa, E. J., Bennett, J. V., Wyatt, c, Pierce, P. E., Olarte, J., Mendoza, P. H., Vasquez. V., Bessudo, D. M. An epidemic-associated episome? J. Infect. Dis. 126:215-218, 1972. 6. Olarte, J., Galindo, E. Salmonella typhi resistant to chloramphenicol, ampicillin and other antimicrobial agents: strains isolated during an extensive typhoid fever epidemic in Mexico. Antimicrob. Agents Chemother. 4: 596-601, 1973. 7. Bessudo, D. M., Olarte, J., Mendoza-Hernandez, P., Galindo, E., Carrillo, J., Gutierrez, Trujillo, G., Kurnate, J. Aislamiento de S. typhi resistente a altas concentraciones de chloranfenicol. Boletin de la Oficinia Sanitaria Panamericana 74: 1-6, 1973. . 8. Overturf, G., Marton, K I., Mathies, A. W., Jr. Antibiotic resistance in typhoid fever. N. Engl. J. Med. 289:463-465, 1973.
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9. Gonzalez-Cortes, A., Bessudo, D., Sanchez-Leyva, R., Fragoso, R., Hinojosa, M., Becerril, P. Waterborne transmission of cloramphenicol-resistant Salmonella typhi in Mexico. Lancet 2:605-607, 1973. 10. Butler, T., Linh, N. N., Ai nold, K, Pollack, M. Chloramphenicol-resistant typhoid fever in Vietnam associated with R factor. Lancet 2: 983-985, 1973. 11. Calderon, E., Gilman, R. H., Snyder, M. J., Vasquez, V., Gomez Barreto, D., Legorreta, J., Rodriguez, R., Martinez, E., Hornick, R. B., Woodward, T. E. A controlled clinical trial of chloramphenicol in chloramphenicol-resistant typhoid fever. Rev. Lat. Am. Microbiol. 16:131-136, 1974. 12. Center for Disease Control. Typhoid fever contracted in Mexico. Morbidity and Mortality Weekly Rep. 21:193-194,1972. 13. Calderon, E. Amoxicillin in the treatment of typhoid fever due to chloramphenicol-resistant Salmonella typhi. J. Infect. Dis. 129(Suppl.) :S219-S222, 1974. 14. Kamat, S. A. Evaluation of therapeutic efficacy of trimethoprim-sulfamethoxazole and chloramphenicol in enteric fever. Br. Med. J. 3: 320-322, 1970. 15. Stamps, T. J., Wicks, A. C. B. Trirnethoprim-sulfamethoxazole (Bactrim) in the treatment of typhoid fever. S. Afr. Med. J. 46: 652-655, 1972. 16. Cardoso, N. Double-blind trial with chloramphenicol and the combination trimethoprim-sulfamethoxazole in typhoid. S. Afr. Med. J. 46: 1286-1287, 1972. 17. Gordon, R. c., Regamey, c., Kirby, W. M. Comparative clinical pharmacology of amoxicillin and ampicillin administered orally. Antimicrob. Agents Chemother. I: 504-507, 1972. 18. Neu, H. c, Winshell, E. B. Pharmacological studies of 6 [D( - )-a-amino-p-hydroxyphenylacetamido] penicillanic acid in humans. Antimicrob. Agents Chemother. 1970: 423-426, 1971. 19. Gilman, R. H., Terrninel, M., Levine, M. M., Hernandez-Mendoza, P., Hornick, R. B. Relative efficacy of blood, urine, rectal swab, bone marrow, and rose-spot cultures for recovery of Salmonella typhi in typhoid fever. Lancet 1:1211-1213,1975. 20. Terminel Valenzuela, N., Mendoza Hernandez, P., Bahena Albarran, I. Tecnica del cultivo de la biopsia de roseola para el diagnostico de la fiebre tifoidea. Rev. Lat. Am. Microbiol. 15:69-70, 1973. 21. Castaneda, M. R. Practical method for routine blood cultures in brucellosis. Proc. Soc. Exp. BioI. Med. 64: I 14-115, 1947. 22. Edwards, P. R., Ewing, W. H. [ed.]. Identification of Enterobacteriaceae. Burgess, Minneapolis, 1962, p. 168-173. 23. Bauer, A. W., Kirby, W. M. M., Sherris, J. c, Turck, M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496, 1966.
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24. Bushby, S. R. Sensitivity testing with trimethoprirnsulfamethoxazole. Med. J. Aust. 1(Suppl.): 10-18, 1973. 25. Bach, M. c, Finland, M., Gold, 0., Wilcox, C. Susceptibility of recently isolated pathogenic bacteria to trimethoprim and sulfamethoxazole separately and combined. J. Infect. Dis. 128(Suppl.): S508-S533, 1973. 26. Neu, H. C. Antimicrobial activity and human pharmacology of amoxicillin. J. Infect. Dis. 129 (Suppl) :SI23-8133, 1974. 27. Snyder, M. J., Perroni, J., Gonzalez, 0., Palomino,
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c., Gonzalez, c., Music, S., DuPont, H. L., Hornick, R. B., Woodward, T. E. Trimethoprim-sulfamethoxazole in the treatment of typhoid and paratyphoid fevers. J. Infect. Dis. 128(Suppl): S734-8737, 1973. 28. Anderson, E. S. Chloramphenicol-resistant Salmonella typhi. Lancet 2: 1494-1495, 1973. 29. Garrod, L. P. Trimethoprim: its possible place in antibacterial therapy. Drugs 1: 3-6, 1971. 30. Kahn, S. B., Fein, S. A., Brodsky, I. Effects of trimethoprim on folate metabolism in man. Clin. Pharmacal. Ther. 9: 550-560, 1968.
