Search

Beginning in mid-2002, a large tuberculosis outbreak occurred among homeless persons in King County, Washington. In order to further monitor the outbreak following its peak in 2003, Mycobacterium tuberculosis isolates from all new King County tuberculosis (TB) patients in 2004 and the first half of 2005 (n = 220) were genotyped by using a rapid comparative genomics-based (genomic deletion-typing) approach, with confirmation by mycobacterial interspersed repetitive units and repetitive-sequence-based PCR (rep-PCR). Results were compared to retrospective genotypic data from 1995 to 2003. The outbreak strain SBRI9, which was not seen among King County homeless persons prior to 2002, accounted for 16 out of 30 TB cases (53%) within this population in 2002. This trend continued with 27 out of 35 cases (77%) caused by the outbreak strain in 2003, 11 out of 13 cases (85%) caused by the outbreak strain in 2004, and 4 out of 10 cases (40%) caused by the outbreak strain in the first 5 months of 2005. Thus, the outbreak strain remained well established within this homeless population throughout the study period. At least four SBRI9 cases were in people who had previously been infected by other strains. The novel PCR-based strain-typing approach used in this investigation proved to be cost-effective and very rapid. In most cases, it was possible to analyze DNA extracted directly from primary isolation (Mycobacterium growth indicator tube) cultures submitted by clinical laboratories, a feature that markedly reduced the delay between diagnosis and strain typing results. This rapid turnaround facilitated public health efforts to prevent new outbreaks involving this strain.

The N and W-Beijing families of Mycobacterium tuberculosis are phylogenetically closely related. The ability of the W-Beijing family to rapidly cause widespread disease is well described; however, few outbreaks involving the N family have been reported outside the New York City, N.Y., area. During 2002 to 2003, Seattle, Wash., experienced a rapidly expanding tuberculosis outbreak involving 38 persons in a 23-month period. The outbreak strain, SBRI9, exhibited the genotypic properties of the N family. Its IS6110 restriction fragment length polymorphism pattern was identical or nearly identical to those of two N family strains that were responsible for clusters of tuberculosis cases, including a large nosocomial outbreak, in New York City and New Jersey from 1989 to 1990. It was also identical to strains involved in late 1990s tuberculosis cases in Michigan, Maryland, and Arkansas. Further monitoring of the N family may show that it shares with the W-Beijing family the propensity to spread rapidly, suggesting that this characteristic evolved prior to the divergence of the two genetic lineages.

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species. DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M. tuberculosis from diverse geographical regions throughout the world. In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed. No mutations were observed in rifampin-susceptible strains. No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M. tuberculosis strains. Drug-resistant M. tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene. However, mutations are not correlated with IS6110 profiling outside of epidemics. The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment. Rifampin-resistant strains of M. leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M. tuberculosis. This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M. tuberculosis.

The assay for serum antibody to the Salmonella typhi capsular polysaccharide (Vi) antigen has recently been revised because of the availability of a purified, highly polymerized Vi antigen. We compared this revised Vi antibody assay to the traditional one for potential usefulness in the surveillance of chronic enteric carriers of S. typhi. The purified Vi antigen of Citrobacter freundii was incorporated into a passive hemagglutination assay for serum Vi antibody; the standard Vi antibody assay was also a hemagglutination assay that employed as the Vi antigen a crude extract of Citrobacter (Ballerup O group 29). As determined by the revised assay, Vi antibody was found in the sera of 22 (71%) of 31 current typhoid carriers, none of 6 resolved carriers, and none of 22 control subjects. According to the traditional assay, Vi antibody was present in 23 of those current carriers (74%), 1 of the resolved carriers (17%), and 4 of the control subjects (18%). The rate of false-positive Vi antibody tests among resolved carriers and control subjects was less with the revised assay (P < 0.05). Successful antimicrobial therapy resulted in a reversion to seronegativity within 1 year in 8 of 10 Vipositive carriers according to the revised assay, but in only 3 of 11 according to the standard assay (P < 0.05). During a 2-year period of observation, 15 (94%) of 16 current typhoid carriers had at least one positive purified Vi antibody test; among 12 of those patients with Vi titers of 1:40 or greater, 9 (75%) were continuously Vi positive. Thus, the revised Vi antibody assay is more specific and no less sensitive than the standard assay for the condition of current enteric carriage of S. typhi. This serological test could be of value in the surveillance of typhoid carriers by public health agencies.

Rosaramicin, a new macrolide antibiotic, was compared with penicillin G in the treatment of pneumococcal meningitis in rabbits. Animals were infected intracisternally with 10(4) colony-forming units of Streptococcus pneumoniae type III (rosaramicin minimal inhibitory/bactericidal concentrations, 0.25/0.5 mug/ml; penicillin G minimal inhibitory/bactericidal concentrations, 0.03/0.06 mug/ml). Treatment was instituted 96 h later. Infusion of rosaramicin at 25 mg/kg per h intravenously for 8 h produced a peak cerebrospinal fluid (CSF) drug concentration of 1.54 mug/ml (range, 0.87-3.6 mug/ml). During this infusion the numbers of pneumococci in CSF decreased from 6.2 +/- 0.5 to 3.36 +/- 1.12 log(10) colony-forming units per ml. Penicillin G, infused at 30 mg/kg per h for 8 h, reached a similar concentration in CSF but caused a greater reduction (P < 0.01) in CSF bacteria, from 6.4 +/- 0.36 to 1.3 +/- 0.67 log(10) colony-forming units per ml. Penicillin G, at 100 mg/kg per day intramuscularly for 5 days, cured 7 of 10 rabbits with pneumococcal meningitis. A higher dose, 300 mg/kg per day for 5 days, was no more efficacious: 11 of 14 rabbits were cured. Rosaramicin at 100 mg/kg per day intramuscularly for 5 days cured only 5 of 15 rabbits with meningitis, but a higher dosage regimen of that drug (250 mg/kg per day intramuscularly) produced acute, fulminant enterocecitis and death within 48 h in seven of eight rabbits. No cytotoxin was detected in the feces of one rabbit with acute enterocecitis. Thus the efficacy of rosaramicin in experimental pneumococcal meningitis, measured by bacterial clearance from CSF and by treatment outcome, was less than that of penicillin G. In addition, high-dose parenteral rosaramicin caused acute, fulminant enterocecitis in a high proportion of treated rabbits.

We studied cefotaxime in the treatment of gonococcal and nongonococcal pelvic inflammatory disease. Cefotaxime was uniformly effective against gonococcal pelvic inflammatory disease. However, 4 of 11 patients with nongonococcal pelvic inflammatory disease had a suboptimal response.

Polymyxin B and rifampin were given to 12 patients with multi-drug-resistant nosocomial Serratia marcescens infections. Eight cures were achieved; drug hepatotoxicity occurred once; one fatal suprainfection was encountered; and two patients died during therapy of causes related to severe underlying illnesses. Polymyxin B and rifampin were uniformly synergistic in vitro against the infecting strains and against 40 additional clinical isolates of S. marcescens.

We studied interactions in vitro between human granulocytes and the yeast-like form of Blastomyces dermatitidis, because granulocytes are prominent in the host response to systemic blastomycosis. In Boyden chamber assays, broth culture filtrates of B. dermatitidis contained levels of granulocyte chemotactic activity that were significantly higher than those present in similar culture filtrates of Histoplasma capsulatum and Cryptococcus neoformans, two fungi that characteristically do not elicit granulocytes in infected tissues. Microscopic study, including electron microscopy, demonstrated that granulocytes phagocytosed B. dermatitidis promptly and efficiently. Moreover, granulocytes emitted light (chemiluminescence) at a brisk rate during phagocytosis of B. dermatitidis, indicating activation of intracellular metabolic pathways. However, fungicidal assay showed that granulocytes (1:1 cell-yeast ratio, 10% serum) killed only 29% of the B. dermatitidis inoculum during 3 h of incubation. Taken together, these findings suggest that there is disparity between phagocytosis and intracellular killing of B. dermatitidis by human granulocytes, perhaps because of resistance of this fungus to granulocyte microbicidal mechanisms.

Eleven children with bacterial meningitis were treated intravenously with amoxicillin sodium to evaluate the efficacy of the parenteral form of amoxicillin for this serious infection and to measure the penetration of the drug into cerebrospinal fluid (CSF). The infecting organisms were Haemophilus influenzae in nine cases and Streptococcus pneumoniae in two. Nine patients had optimal responses to amoxicillin sodium, 200 mg/kg per day for 14 days. Bacteria were also eradicated from CSF of the other two, but one experienced fever and culture-negative CSF pleocytosis after cessation of amoxicillin, and the other developed H. influenzae empyema 2 weeks after termination of therapy. By comparison, 7 of 10 children with meningitis responded optimally to ampicillin (nonrandomized design) during the period of study. The mean peak CSF concentration of amoxicillin was 3.14 mug/ml (ca. 7% of the concomitant mean peak serum level) early during therapy. However, meningeal penetration of the drug declined to a mean peak of 0.63 mug/ml on the final day of therapy. Mild transient neutropenia, noted in five patients, was the most common side effect of amoxicillin sodium therapy; five patients treated with ampicillin also experienced reversible neutropenia. Thus, intravenous amoxicillin sodium provided therapy for bacterial meningitis comparable to that of ampicillin in this limited case-control study.

Antibiotic-associated pseudomembranous colitis has been linked with Clostridium difficile toxin. We examined the effect of toxins from four strains of C. difficile isolated from patients with pseudomembranous colitis on colonic adenylate (EC 4.6.1.1) and guanylate cyclase (EC 4.6.1.2) activities. Partially purified toxins had a cytotoxic effect on hamster fibroblasts in culture at a concentration of 10 ng/ml. Likewise, these toxins enhanced colonic guanylate cyclase activity two- to threefold, with the maximal stimulation being at 10 ng/ml. These toxins also enhanced guanylate cyclase activity in ileum, cecum, and duodenum. Both the cytotoxic activity on hamster fibroblasts and the enhancement of hamster guanylate cyclase activity were inhibited by antiserum to C. difficile toxin. These same toxins inhibited adenylate cyclase activity at a 100-ng/ml concentration, but had no effect at 10 ng/ml. They also had no effect at any concentration on colonic Na+-K+ adenosine triphosphatase. To be sure that the findings were not due to a contaminant, a purified C. difficile cytotoxin was used, and the same findings were found with the pure cytotoxin (at a 100-fold-lower concentration). The data suggest that activation of guanylate cyclase may be a factor in the pathogenesis of antimicrobial-associated pseudomembranous colitis.