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To characterize IgM and IgG antibody responses in Rocky Mountain spotted fever (RMSF), a microtiter enzyme-linked immunosorbent assay (ELISA) using density gradient-purified Rickettsia rickettsii as antigen was developed. Sera of vaccinated individuals and patients with RMSF were tested by ELISA and by indirect fluorescent antibody (IFA) tests. Diagnostic agreement between ELISA and the IFA test was 76% and 52% for IgG and IgM antibody, respectively. Diagnostic agreement between the ELISA for IgG antibody and the IFA test for total immunoglobulins was 84%. The ELISAs for IgM and IgG antibody were as specific (100%) and as sensitive (100%0) as the IFA test (83o%-100%o) in detecting antibody increases in paired sera from persons with RMSF and were superior to the IFA test in detecting seroconversions in vaccinees. The ELISA also detected antibodies in a single convalescent-phase serum with sensitivity and reliability. The ELISA for IgG antibody is appropriate for seroepidemiology and serodiagnosis since it permits measurement of antibody at a single dilution of serum up to a year after illness.

Bacteria have demonstrated a disconcerting ability to develop resistance to antimicrobial agents nearly as quickly as new compounds become available. During the past two decades the molecular bases of several types of resistance have been elucidated. Mechanisms of resistance include the transference of genetic material either through conjugation (involving direct contact between microorganisms), or indirectly through transduction (involving bacteriophages). In addition to this "infectious" drug resistance, genetic mutations which permit the utilization of new metabolic pathways, and the production of enzymes which can inactivate the antimicrobic have been described. One particularly complex problem has been the ability of many Enterobacteriaceae to develop resistance to multiple antimicrobials simultaneously. The possible effect of such an occurrence is illustrated by the recent epidemic of multiply resistant Salmonella typhi in Mexico. Because the typhoid bacilli shared an identical resistance pattern to an epidemic Shigella dysenteriae type 1 the in vivo interspecies transmission of resistance has been postulated. Understanding the various mechanisms of resistance development should allow more rational use of antimicrobial agents.

Minimum inhibitory concentrations (MIC) of pirbenicillin against 135 clinical isolates of Pseudomonas aeruginosa were one-fourth of those required for carbenicillin but two times higher than those for BL-P1654. Increasing the inoculum size produced an adverse effect on the bactericidal activity for all three antibiotics. This was more apparent for pirbenicillin than for carbenicillin, but less than the effect on BL-P1654. When concentrations of antibiotics likely to be achieved clinically were used, gentamicin increased the inhibitory and bactericidal effects of all three semisynthetic penicillins for the majority of isolates. Strains highly resistant to the aminoglycoside antibiotic, however, were inhibited no more by the penicillin-gentamicin combinations than by the most effective of the antibiotics alone.

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 typhi. 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 ampicillin-amoxicillin-resistant, typhoid fever.

Rats were made neutropenic by intraperitoneal (ip) injection of cyclophosphamide. Those neutropenic (mean white blood cell count of 470/mm3) rats were challenged intraperitoneally with Pseudomonas aeruginosa to assess the efficacy of single agent therapy with either imipenem, latamoxef (moxalactam) or amikacin, or combination therapy with imipenem-amikacin or latamoxef (moxalactam)--amikacin. Pharmacokinetic studies were performed in rats to assure that therapy was equivalent during therapeutic trials. Three levels of bacterial challenge (4 LD50, 13 LD50 and 250 LD50) were examined. At all challenge levels, single agent therapy with latamoxef (moxalactam) failed to significantly protect rats from fatal bacteraemia. Single-agent therapy with amikacin did significantly protect rats from fatal bacteraemia at the lower challenge levels, but not at the 250 LD50 challenge. Single agent therapy with imipenem significantly protected rats at all challenge. Single agent therapy with imipenem significantly protected rats at all challenge levels. In-vitro studies established a synergistic effect when combination antibiotics were used. This correlated with in-vivo findings that combination therapy resulted in improved rat survival and recovery of fewer Ps. aeruginosa isolates. The latamoxef (moxalactam)-amikacin combination was more effective than either agent alone, but was not more effective than imipenem alone. The imipenem-amikacin combination was the most effective therapeutic regimen tested. These results suggest that imipenem alone, and particularly when combined with an aminoglycoside, is effective in treating serious Ps. aeruginosa infections in neutropenic rats. Clinical studies in infected immunocompromised patients may be warranted.

Four studies were done with streptomycin-dependent Salmonella typhi as an oral, attenuated vaccine. Studies 1 and 3 employed freshly harvested vaccine, whereas studies 2 and 4 involved lyophilized vaccine. Five to eight doses (3 x 10(10)-10(11) organisms/dose) were given; oral streptomycin (1.0 g) was administered concomitantly in studies 2 and 3, with only two of the doses of vaccine in study 1, and was not given in study 4. No adverse reactions were encountered in 179 vaccinated men, and 94% of the men excreted the vaccine. In challenge studies (which included the control groups) with 10(5) virulent S. typhi organisms (Quailes strain), the fresh vaccine was highly protective (66%-78% efficacy), while lyophilized vaccine gave no clinical protection. Fresh vaccine also interfered significantly with intestinal proliferation of virulent S. typhi; only 17% of the vaccinees excreted organisms as compared with 75% of the controls. Studies of protection in mice showed no difference between immunogen content of the fresh and the lyophilized vaccines. Field trials with streptomycin-dependent, oral typhoid vaccine must await development of a lyophilized product that will retain the protective properties of the vaccine.

A mutant (Ty21a) of Salmonella typhi, which lacks the enzyme uridine 5'-diphosphate-glucose-4-epimerase, was evaluated in volunteers for use as a live attenuated oral typhoid vaccine. Five to eight doses of vaccine (containing 3-10(10) viable organisms per dose) were given to 155 men without significant side effects. The rate of excretion of the vaccine strain in stools was low, and the majority of isolations occurred on day 1 after vaccination. Revertants able to fement galactose were not found in any of 958 stool isolates tested. The mutant, strain Ty21a, grown in brain-heart infusion broth (BHIB) with 0.1% galactose, produces more O side chain than the same vaccine strain cultivated without galactose. Volunteers vaccinated with strain Ty21a grown in galactose and then challenged with 10(5) virulen S. typhi were significantly protected from disease and also had decreased stool carriage of S. typhi as compared with controls. Strain Ty21a grown without galactose did not provide vaccinees significant protection nor decrease fecal excretion of S. typhi as compared with controls. Strain Ty21a, when grown in BHIB with 0.1% galactose, results in a safe, stable and protective oral vaccine that warrants further study in field trials.

In an attempt to develop a safe, proliferating, oral, attenuated vaccine against shigellosis, genes that control the synthesis of group- and type-specific somatic antigens of Shigella flexneri 2a were transferred via conjugation to a recipient strain of Escherichia coli. The resultant hybrid (E. coli expressing shigella surface antigens) vaccine strain, PGAI 42-1-15, believed to have a complete (smooth) lipopolysaccharide, was given to volunteers in two vaccination-challenge studies. The vaccine was well tolerated and gave evidence of intestinal proliferation. In trial no. 1, volunteers given two doses of vaccine one month apart were challenged after eight weeks with 10(4) virulent S. flexneri 2a. Attack rates were comparable in vaccinees (50%) and controls (40%). In trial no. 2, vaccinees were given three weekly doses of vaccine and were challenged four weeks later with a small inoculum (10(2)) of S. flexneri 2a. Again, attack rates among vaccinees (47%) and controls (39%) were similar. It is unclear why this theoretically ideal, live shigella vaccine failed to protect against S. flexneri 2a.

Two Escherichia coli strains isolated in Vietnam from American soldiers with diarrhea and acute "colitis" were examined for virulence in both in vitro and in vivo experimental models. Their biologic properties were compared with those of an enterotoxin-producing Esch. coli strain implicated in disease of swine as well as with those of four other Esch. coli and a virulent shigella strain. The two Vietnam strains produced an enterotoxin in a rabbit ileal-loop model and in volunteers caused a diarrheal syndrome resembling that of cholera. Two nontoxigenic Esch. coli strains gave evidence of penetrating epithelial cells in laboratory models and caused a shigella-like illness in man characterized by dysentery, tenesmus, urgency, hyperpyrexia and hypotension with systemic toxemia. Thus, strains of Esch. coli can cause disease in man by at least two mechanisms: elaboration of a cholera-like enterotoxin; and shigella-like intestinal epithelial penetration.

Summary Viable rickettsiae were isolated from the lymphatic tissues of a patient one year following recovery from Rocky Mountain spotted fever. The recovered agent was maintained by serial passage in guinea pigs and fertile hens' eggs. Identity of the isolated agent was established by: The development of specific complement fixing antibodies in guinea pigs.Direct visualization of the microorganisms in yolk sac tissues.The demonstration of its ability to afford cross immunity to a standard laboratory strain of R. rickettsii.

Myron M. Levine, Herbert L. DuPont, Samuel B. Formal, Richard B. Hornick, Akio Takeuchi, Eugene J. Gangarosa, Merrill J. Snyder, and Joseph P. Libonati From the Shigella Vaccine Development Program, University of Maryland School of Medicine, Baltimore, Maryland; The Walter Reed Army Institute of Research, Washington, D.C.; and the Bacterial Diseases Branch, Epidemiology Program, Center for Disease Control, Atlanta, Georgia

ConclusionSerological studies on convalescent specimens from cases of Rocky Mountain spotted fever show the presence of epidemic and murine agglutinins and epidemic neutralizing antibodies although there is no evidence of cross complement fixation. This evidence fits in with the observations of Castaneda and Silva and those of Parker, and indicates that there is a serologic relationship between epidemic typhus fever and Rocky Mountain spotted fever. The data presented in this paper must be considered in evaluating the results obtained with rickettsial agglutination or the neutralizing antibody when these tests are used as diagnostic procedures.

In the United States, epidemic shigellosis has been a particularly important problem in confined groups such as the military, institutions for the mentally retarded, and Indian reservations. It is in such confined populations that an effective Shigella vaccine would be most beneficial. Shigella flexneri 2a is an important cause of both sporadic and epidemic shigellosis in the United States. Between the years 1963 and 1966 this pathogen accounted for approximately one-fourth of all Shigella isolates reported to the National Communicable Disease Center [1]. In 1967 this center recorded 11,405 isolations of all Shigella [2]. In order to test the effectiveness of oral attenuated living S. flexneri 2a vaccines, an experimental model has been developed in man. The response of volunteers to oral challenge by virulent S. flexneri 2a was determined at doses of 104 -108 viable organisms.

A trial was conducted in naturally-occurring disease to compare the efficacy of treatment with trimethoprim-sulfamethoxazole (TMP-SMZ) with oral chloramphenicol. The majority of patients treated with TMP-SMZ (79%) responded just as well as did those treated with oral chloramphenicol, as measured by time of disappearance of fever after therapy. Seven patients treated with TMP-SMZ, however, failed to respond after 11 or more days of treatment. Neither levels of drug in serum nor in vitro sensitivity tests of the isolated Salmonella could account for the slow response of these patients to TMP-SMZ. Enteric fever is a major medical problem both in the undiminished numbers of cases occurring each year and in geographic distribution not only in the developing but in the developed countries. In addition, although specific antimicrobial agents are available, the therapeutic response to these drugs and the risk of side effects still leave the hope for more effective agents. The spectrum of disease, from mild and self-limiting to severe, makes evaluation of therapy in enteric fever difficult. Early reports of the efficacy of trimethoprim-sulfamethoxazole (TMP-SMZ) encouraged us to include this combination in a therapeutic trial of randomly assigned drugs in the treatment of naturally-occurring typhoid fever.

We have evaluated the utility of optimal sampling strategy coupled with adaptive study design in the determination of individual patient and population pharmacokinetic parameter values. In 9 patients with cystic fibrosis receiving a short (1 minute) infusion of ceftazidime pharmacokinetic parameter values were determined with a nonlinear least-squares estimator analyzing a traditional, geometrically spaced set of 12 postinfusion serum samples drawn over 8 hours. These values were compared with values generated from four sample subsets of the 12 obtained at optimal times and analyzed by nonlinear least-squares estimator, as well as a maximum a posteriori probability Bayesian estimator with prior distributions placed on beta and clearance. The four sampling times were determined according to an adaptive design optimization technique that employs sequential updating of population prior distributions on parameter values. Compared with the 12-point determination, the four optimal points analyzed with the maximum a posteriori probability Bayesian estimator faithfully reproduced both microscopic and hybrid pharmacokinetic parameter values for individual patients and, consequently, also produced accurate measures of population central tendency and dispersion. This has important implications in being able to more efficiently derive target patient population pharmacokinetic information for new drugs. This should also allow generation of better concentration-effect relationships in populations of interest.

The spectrum of illness and the immunologic response produced by cholera in volunteers were studied. The strains of Vibrio cholerae used were classical Inaba 569B and classical Ogawa 395. An oral dose of 108 organisms in buffered saline was required to induce the diarrhea of cholera. When given with live organisms, NaHCO3 lowered the infecting dose from 108 to 104 organisms. Clinical manifestations of infection varied from culturally positive formed stools to "rice water" diarrhea. Severe diarrhea did not have an explosive onset but rather progressively increased in volume during a 24-hr period. In 45% of cases the stool was positive for V. cholerae before the onset of diarrhea. Titers of vibriocidal antibody rose after diarrhea, peaked the second week after challenge, and rapidly fell during the next four weeks.

In May and June 1973, 11 patients on the surgical service at the University of Maryland Hospital had bacteremia caused by Pseudomonas species. Seven of the isolates recovered from blood cultures had the same antibiogram (sensitive only to chloramphenicol and tetracycline). Ten of the 11 patients were given 25% normal serum albumin (human) shortly before the onset of symptoms. In contrast, only two of seven patients with bacteremia due to Psuedomonas aeruginosa in May and June (P =0.013) and only nine of 20 patients located in surgical special care units during these months (P =0.014) were given this product. When cultured, the albumin in one of 54 previously unopened vials from the implicated lot yielded Pseudomonas cepacia sensitive only to chloramphenicol, tetracycline, and nalidixic acid. Subsequent investigation showed that five more patients in four other hospitals had symptoms of bacteremia shortly after the infusion of different lots of albumin from the same manufacturer, and in four cases P. cepacia was cultured from the suspect albumin. Since sterility testing by manufacturers may not detect low-frequency contamination, surveillance of nosocomial infections, investigation of unusual disease clusters, and prompt reporting of suspect reactions are essential in the control of such outbreaks.

Introduction Alteration of man's response to measles infection by parenteral administration of serum containing specific antibodies was first described by Nicolle and Conseil.1Since 1918, several reports have confirmed the effectiveness of convalescent measles serum in the prevention of this disease as well as its usefulness in the therapy of measles during the preeruptive phase.2-5Placental extracts were found to be at least as effective as human convalescent serum in aborting measles.6Similarly, the concentration of antibody from normal human serum in Cohn's Fraction II, composed primarily of gamma globulin,7yielded a product with potent measles-preventive activity. Evaluation of the protective and attenuating action of human gamma globulin in several large groups of susceptible children exposed to measles revealed the effectiveness of concentrated antibody.8,9Furthermore, it was proposed on the basis of these studies that modification of measles could best be accomplished if globulin were

Age Distribution of Measles Antibody A survey of 900 serum specimens from residents of Baltimore, representing all age groups, demonstrated that the acquisition of complement fixing antibodies for measles was most rapid in the second to third and sixth years of life (Figure). Between the 10th and 15th years, the incidence of measles complement fixing antibody reached a maximum of 80% to 90%, which incidence persisted for all ages above 15 years. Geometric mean titers of those positive in each age group were strikingly similar. In all our studies, sex differences were not observed. The age distribution of antibodies was in agreement with previous findings determined by historical methods. 1 It is to be noted that sera from adults who did not possess complement fixing antibody were invariably positive when tested by tissue culture neutralization. Why in most cases measles complement fixing antibody was retained for life without diminution of

Introduction Standardization of live, attenuated measles vaccine will be influenced by the relationship between the amount of virus administered and host response as well as by stability of the commercial product. Optimal dosage of virus may be defined as the number of infective units producing maximal immune response and minimal overt reactivity. In an effort to measure sensitivity of the human host to parenterally inoculated measles virus vaccine and to assess the effect of varying dosage on reaction rate and immunogenicity, titration of 2 measles vaccines in susceptible children was carried out. In addition, sensitivity of the respiratory tract has been measured indirectly. These data will be presented in the following report. Methods Children proven by serologic tests to be susceptible to measles were given 1.0 ml. quantities of serial 10-fold dilutions of measles vaccine prepared in medium 199 immediately prior to inoculation. The canine renal and chick embryo cell

Introduction Control of measles through active immunization has been the object of numerous studies. Measles first was transmitted artificially to susceptible human beings by Home,1who exposed scarified skin of volunteers to blood from patients in the acute phase of this disease. In a similar attempt to produce active immunity, Hektoen2injected fresh infective blood subcutaneously into susceptible persons who subsequently developed measles. Failure to produce attenuated disease apparently discouraged further attempts to induce active immunity against measles. Adaptation of measles virus to growth in embryonated hen's egg and the subsequent use of this virus as an immunizing agent was reported by Rake and his associates between 1939 and 1943.3-8Virus passaged from 4 to 108 times via the chorioallantoic route did not induce in susceptible children a significant degree of protection from naturally occurring measles,9and interest in this problem again waned. In 1954, Enders