Direct antimicrobial drug susceptibility testing of Mycobacterium tuberculosis by the radiometric method.

Direct-drug-susceptibility tests were performed on clinical specimens positive for acid-fast bacilli by either Ziehl-Neelsen or fluorochrome staining. The results of conventional agar dilution (Vestal, 1975) and a modified radiometric (BACTEC) method were compared. A total of 580 smear-positive specimens were tested by the BACTEC method at three separate sites. Three hundred and seventy-seven of these were culture positive for M. tuberculosis, and 343 (91%) yielded acceptable direct-susceptibility-test results. We used the conventional method to determine that 343 of 519 smear-positive specimens were culture positive for M. tuberculosis, and 212 (62%) produced acceptable results within 3 wks. Conventional results were reported in 3-4 wks, while the time required to obtain results with the BACTEC method ranged from 5 to 21 days (average 11.5 days). Results indicate that the radiometric method provides reportable results more frequently with time savings as compared to the conventional method.

Immunity of cholera in man: relative role of antibacterial versus antitoxic immunity.

Purified cholera toxoid is antigenic when given enterally and orally. Purified toxoid fails to provide protection against experimental challenge. Clinical cholera confers formidable protection against homologous or heterologous rechallenge. Failure to culture vibrios from intestinal fluid or stool of re-challenge volunteers suggests that the predominant immune mechanism is antibacterial rather than antitoxic.

Evaluation of rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis.

A total of 106 isolates of Mycobacterium tuberculosis were tested for drug susceptibility by the conventional 7H11 plate method and by a new rapid radiometric method using special 7H12 liquid medium with 14C-labeled substrate. Results obtained by the two methods were compared for rapidity, sensitivity, and specificity of the new test method. There was 98% overall agreement between the results obtained by the two methods. Of a total of 424 drug tests, only 8 drug results did not agree, mostly in the case of streptomycin. This new procedure was found to be rapid, with 87% of the tests results reportable within 4 days and 98% reportable within 5 days as compared to the usual 3 weeks required with the conventional indirect susceptibility test method. The results of this preliminary study indicate that the rapid radiometric method seems to have the potential for routine laboratory use and merits further investigations.

Gonorrhea prevalence in the Maryland state prisons.

During a 6-month period from July 1 through December 31, 1986, 2,598 consecutive men were screened for gonorrhea upon entry into the Maryland Division of Corrections. Twenty-eight men (1.1%) demonstrated positive cultures for gonorrhea with 27 of them (92%) asymptomatic. This prevalence is substantially lower than other rates reported from prisons and detention centers. Although the role of asymptomatic males as a reservoir for gonorrhea has been well delineated, standard recommendations for screening high-risk male populations such as prison inmates have not been established. These data suggest that screening in some correctional facilities may not be cost-effective given the low prevalence observed. In addition, contact tracing may be delayed by long holding periods in local detention facilities prior to transfer to the state prison system. These data underscore the need for additional analysis to determine the most effective program structure for screening in correctional facilities and detention centers.

Epidemiology and spectrum of Vibrio infections in a Chesapeake Bay community.

The clinical and epidemiologic features of vibrio infections occurring over 15 y in a hospital adjacent to the Chesapeake Bay were examined. Microbiologic techniques included the use of thiosulfate citrate bile salts sucrose (TCBS) medium on all stool specimens. Among 40 vibrio isolates from 32 patients, eight species were identified: V. parahaemolyticus (16), V. vulnificus (10), non-O1 V. cholerae (7), V. mimicus (3), V. hollisae (1), V. fluvialis (1), V. alginolyticus (1), and an unnamed halophilic vibrio. Sources included stool (13), wound (11), sputum (3), ear (3), bone (1), and gallbladder (1). While illnesses were diverse, they were relatively mild and self-limited with no mortality. The mild clinical spectrum and especially lack of mortality are in sharp contrast to the findings of previous reports. Even with the proximity to the bay and use of optimal isolation techniques, the demonstrated incidence of vibrio infections was low (1.6/100,000/y). The routine use of TCBS medium, which has been advocated for high-risk areas, was not cost-effective.

Efficacy of a live oral cholera vaccine in human volunteers.

A live attenuated oral cholera vaccine has been given to volunteers with complete safety. The vaccine strain appeared to multiply significantly in intestinal contents and to result in appreciable vibriocidal antibody response. Subsequent challenge with virulent Inaba vibrios demonstrated substantial protection from clinical illness and from vibrio shedding. The possibility of reversion to toxin production is discussed.

Live oral cholera vaccine: evaluation of the clinical effectiveness of two strains in humans.

El Tor Ogawa C14-S5 and EW-6, two live vaccine candidate strains, were given to volunteers in varying doses with and without bicarbonate. Vibrios were found in the stool of one of 32 men given the vaccine strain, and only three men developed a significant titer rise (fourfold or greater) at 2 weeks of vibriocidal or antitoxic antibody. Five men who had previously received 10(9) organisms of the C14-S5 strain were challenged subsequently with virulent Ogawa 395 Vibrio cholerae. The rate of clinical infection in these men was no different than in unvaccinated controls. It was demonstrated that the live oral cholera vaccines did not remain viable in the intestine long enough to act antigenically.

Serotype distribution of invasive group B streptococcal isolates in Maryland: implications for vaccine formulation. Maryland Emerging Infections Program.

Invasive group B streptococcal (GBS) infection is a major health problem among infants and adults. The formulation of GBS vaccines depends on knowledge of the GBS serotype distribution. Serotype V GBS infection appears to have recently emerged, suggesting that the serotype distribution changes over time. GBS isolates from 210 pediatric patients, 23 pregnant women, and 314 nonpregnant adults with invasive infection in Maryland were studied. The predominant serotypes from infants with early-onset disease were as follows: serotype III, 38% of isolates; serotype Ia, 36%; serotype V, 13%; and serotype II, 11%. Although the majority (60%) of isolates among infants with late-onset infection were serotype III, serotype Ia (23%) was also common. The predominant serotype among isolates from nonpregnant adult patients was serotype V, accounting for 29% of the isolates. The serotype distribution differs between pediatric patients and adults and is changing over time. The inclusion of a relatively small number of serotypes in a GBS vaccine could provide protection against the vast majority of isolates.

Role of solid media when used in conjunction with the BACTEC system for mycobacterial isolation and identification.

This study evaluated the necessity and the contribution of solid media when used in conjunction with radiometric Middlebrook 7H12 (BACTEC 12B; Becton Dickinson, Towson, Md.) medium for recovery and complete identification of mycobacteria. Each of 1,184 digested, decontaminated respiratory specimens was inoculated into one BACTEC 12B vial, one 7H11 plate, and two Lowenstein-Jensen (LJ) slants. When the 12B vial was smear positive for acid-fast bacilli, the organisms were subcultured onto LJ slants and the BACTEC p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test system was inoculated with the organisms. Niacin tests were performed by using the growth from the original LJ slants and organisms from the LJ slants subcultured from 12B or 7H11 medium. The times to achieve definitive NAP and niacin test results were recorded. Recovery of all 143 isolates found in this study could not be achieved with a single medium. Among the three media, the highest percentage (92.8%) of Mycobacterium tuberculosis isolate recovered was with BACTEC 12B. The use of either 7H11 medium or LJ slants along with a 12B vial increased by 4 to 6% the total percentage of M. tuberculosis organisms that were isolated. Isolation of the M. tuberculosis complex and NAP differentiation in 12B medium were completed in an average of 17 days. On average, isolation and definitive niacin test results for M. tuberculosis cultures were obtained in 39.3 days by a conventional procedure and in 36.3 days when 12B subcultures were used. These results support the conclusion that LJ slants contribute 4 to 6% increased recovery of M. tuberculosis when used in conjunction with 12B medium. Additionally, a subculture onto LJ slants from 12B medium yielded sufficient growth for niacin testing earlier than an original LJ slant did.

Molecular epidemiology of neonatal meningitis due to Citrobacter diversus: a study of isolates from hospitals in Maryland.

Six cases of neonatal meningitis due to Citrobacter diversus were diagnosed in three Baltimore (Maryland) hospitals between 1983 and 1985. Using plasmid profiles, biotypes, serotypes, and chromosomal restriction endonuclease digests as epidemiological markers, we studied 63 isolates of C. diversus (including four isolates from cerebrospinal fluid) from these and seven other hospitals in Maryland. Within two of the three hospitals with meningitis cases, the same strain of C. diversus was isolated from case infant(s), healthy neonates, and nursery personnel. In all three hospitals, C. diversus strains different from those implicated as a cause of meningitis were also isolated. Other than the meningitis-associated strains, 15 different strains of C. diversus were isolated from infants in the hospitals studied, with several distinct clusters of asymptomatic, colonized infants identified.

Persistence of cholera in the United States: isolation of Vibrio cholerae O1 from a patient with diarrhea in Maryland.

A case of cholera was identified in Baltimore County, Md., in October 1984. The Vibrio cholerae O1 isolate from the patient was hemolytic, biotype El Tor, serotype Inaba, and was toxigenic by the Y-1 adrenal cell assay; on Southern blot analysis, the strain had a unique HindIII restriction site in the cholera toxin gene identical to that of other U.S. V. cholerae O1 isolates. Two days before he became ill, the patient had eaten meat from crabs harvested along the Texas coast.

Investigation of an outbreak of Salmonella enteritidis gastroenteritis associated with consumption of eggs in a restaurant chain in Maryland.

Salmonella enteritidis ser. enteritidis was isolated from patrons and employees of three restaurants in a restaurant chain in Maryland during August and September 1985. Isolates from all three restaurants had identical plasmid profiles; this profile was present in 13 of 40 randomly selected S. enteritidis isolates received by the Maryland state health department laboratory during a comparable time period. The outbreak in one restaurant resulted in at least 71 illnesses, with 17 persons known to have been hospitalized. Scrambled eggs served on a "breakfast bar" were implicated as the vehicle of transmission in this restaurant, with eggs a possible vehicle in another of the three restaurants. The data point out the risks associated with improper handling of eggs in food service establishments, provide further evidence for the observed association between S. enteritidis and eggs in the northeastern United States, and demonstrate the utility of plasmid analysis in investigation of outbreaks involving common Salmonella serotypes.

Rapid broth macrodilution method for determination of MICs for Mycobacterium avium isolates.

A multicenter study was done to investigate the accuracy and reproducibility of a method for determining the MICs of antimicrobial agents against the Mycobacterium avium complex in 7H12 broth with the BACTEC system. In phase I, with eight drugs and 10 strains, intralaboratory reproducibility was 95.7 to 100%, allowing a 1-dilution difference upon repeat testing. The results of phase II testing with 41 additional strains were consistent with those obtained in phase I, with good interlaboratory reproducibility. The radiometric method was validated by sampling and plating of the same broth cultures and determining, by the number of CFU per milliliter, the lowest drug concentration that inhibited more than 99% of the initial bacterial population. Three test concentrations of each drug and the tentative interpretation of results are proposed. Radiometric MIC determination has the potential to become the method of choice for clinical microbiology laboratories and evaluation of new agents for the treatment of M. avium infections, both pulmonary and disseminated.

Changing clonal patterns of Salmonella enteritidis in Maryland: evaluation of strains isolated between 1985 and 1990.

We examined isolates from 203 sporadic and outbreak-associated Salmonella enteritidis cases occurring in Maryland between 1985 and 1990. Plasmid profiles were determined for all isolates; 52 isolates were phage typed. Ten plasmid profiles were identified. A single profile (consisting of a single ca. 55-kb plasmid) emerged as the predominant profile in Maryland during the study period. This profile (which was closely associated with phage type 8) accounted for 86% of a group of isolates from sporadic cases in 1988 and 1989, compared with 43% of the 1985 isolates. Strains with this profile were identified in four of nine outbreaks, including one of three outbreaks in which eggs were implicated as a vehicle. While plasmid profiles and phage typing appear to provide complementary means of identifying specific strains of S. enteritidis, the emergence of what appears to be a single predominant clone has reduced the discriminant ability of both typing systems. The factors be a single predominant clone has reduced the discriminant ability of both typing systems. The factors contributing to the emergence of this one clone are still not well understood.

Clostridium perfringens food poisoning: use of serotyping in an outbreak setting.

An outbreak of Clostridium perfringens food poisoning occurred among attendees of a firehouse luncheon. The predominant symptoms of diarrhea (100%) and abdominal pain (81%) among case-patients, the mean incubation period (13.4 h), and the mean duration of illness (21.2 h) were all characteristic of C. perfringens enteritis. Roast beef, although not epidemiologically implicated, was the most likely vehicle of transmission. Fecal specimens from case-patients contained a median C. perfringens spore count of greater than 10(6) and yielded isolates that were heat sensitive and predominantly nonhemolytic, produced C. perfringens enterotoxin A, and, in the majority of specimens (four of five), were identical in serotype. Food samples were negative. This outbreak demonstrates that following enumeration of C. perfringens from a suitable number of fecal specimens from case-patients, serotyping of the isolates may be helpful in implicating C. perfringens as the cause of foodborne illness. This is especially true when implicated food items test negative or are no longer available for testing.

Relapsing invasive group B streptococcal infection in adults.

OBJECTIVE: To study recurrent group B streptococcal infection in adults. DESIGN: Patients with more than one reported group B streptococcal infection were identified through active surveillance for this infection. Sterile-site group B streptococcal isolates were evaluated for serotype and molecular subtyping using restriction endonuclease analysis of chromosomal DNA (REAC). SETTING: All acute-care hospitals in Maryland. PATIENTS: Nonpregnant residents of Maryland 18 years of age or older. RESULTS: 22 adults had at least two group B streptococcal episodes that were separated by 2 to 95 weeks (mean, 24 weeks). Of 395 patients with invasive group B streptococcal infection who survived the first episode and were followed for at least 1 year, 17 (4.3% [95% CI, 2.6% to 6.9%]) had more than one episode. Several patients were found to have endocarditis or osteomyelitis during the second episode. Group B streptococcal isolates from both episodes were obtained from 18 of 22 patients. Of the 18 isolate pairs, 13 (72% [CI, 46% to 90%]) had identical REAC patterns; the probability that at least 13 matches would be found by chance alone was less than 0.000001. Among patients with recurrent infection caused by the same strain, the interval between episodes was shorter (mean, 14 weeks) than that among patients with recurrent infection caused by another strain (mean, 43 weeks; P = 0.05). CONCLUSIONS: Recurrent group B streptococcal infection is common among adults and in most cases appears to be caused by relapse. The optimal management of adults with a first episode of group B streptococcal infection needs to be further defined to minimize the likelihood of recurrent disease.

Attenuated, streptomycin-dependent Salmonella typhi oral vaccine: potential deleterious effects of lyophilization.

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.

Evaluation of a UDP-glucose-4-epimeraseless mutant of Salmonella typhi as a liver oral 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.