Epidemic Leptospirosis Associated with Pulmonary Hemorrhage—Nicaragua, 1995

In October 1995, epidemic “hemorrhagic fever,” without jaundice or renal manifestations, was reported in rural Nicaragua following heavy flooding; 2259 residents were evaluated for nonmalarial febrile illnesses (cumulative incidence, 6.1%) and 15 (0.7%) died with pulmonary hemorrhage. A case-control study found that case-patients were more likely than controls to have ever walked in creeks (matched odds ratio [MOR], 15.0; 95% confidence interval [CI], 1.7–132.3), have household rodents (MOR, 10.4; 95% CI, 1.1–97.1), or own dogs with titers ≥ 400 to Leptospira species (MOR, 23.4; 95% CI, 3.6–`). Twenty-six of 51 case-patients had serologic or postmortem evidence of acute leptospirosis. Leptospira species were isolated from case-patients and potential animal reservoirs. This leptospirosis epidemic likely resulted from exposure to flood waters contaminated by urine from infected animals, particularly dogs. Leptospirosis should be included in the differential diagnosis for nonmalarial febrile illness, particularly during periods of flooding or when pulmonary hemorrhage occurs

Two-Component Direct Fluorescent-Antibody Assay for Rapid Identification of Bacillus anthracis

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non–B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens

Antimicrobial Susceptibility Testing of Bacillus anthracis: Comparison of Results Obtained by Using the National Committee for Clinical Laboratory Standards Broth Microdilution Reference and Etest Agar Gradient Diffusion Methods

We determined the patterns of antimicrobial susceptibility of 65 isolates of Bacillus anthracis (50 historical and 15 recent U.S. clinical isolates) to nine antimicrobial agents using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution reference method. The results for the 50 historical B. anthracis isolates obtained by the broth microdilution method were compared to those generated by the Etest agar gradient diffusion method. One isolate of B. anthracis was β-lactamase positive and resistant to penicillin (MIC, 128 μg/ml); a second isolate, which was β-lactamase negative, was borderline penicillin resistant, with the penicillin MICs for the isolate varying from 0.12 to 0.25 μg/ml; and the remainder of the isolates were β-lactamase negative and penicillin susceptible (MICs, ≤0.12 μg/ml). Approximately 78% of the isolates showed reduced susceptibility to ceftriaxone (MICs, ≥16 μg/ml). All B. anthracis isolates were susceptible to chloramphenicol (MICs, ≤8 μg/ml), ciprofloxacin (MICs, ≤ 1 μg/ml), clindamycin (MICs, ≤0.5 μg/ml), rifampin (MICs, ≤0.5 μg/ml), tetracycline (MICs, ≤0.06 μg/ml), and vancomycin (MICs, ≤2 μg/ml) by use of NCCLS breakpoints for staphylococci. All 15 recent B. anthracis isolates from the United States were susceptible to penicillin, doxycycline, and ciprofloxacin. By use of the susceptibility breakpoint for staphylococci of ≤0.5 μg/ml, 97% of the B. anthracis isolates tested would have been categorized as intermediate to erythromycin. No statistically significant difference was found between the results of broth microdilution testing and the results of the Etest method for any of the antimicrobial agents tested; however, the results for penicillin obtained by the Etest were 1 to 9 dilutions lower than those obtained by the broth microdilution method. The differences in the penicillin MICs by the Etest method and the difficulties of reading the Etest results through the glass of a biological safety cabinet may limit the utility of this alternate susceptibility testing method for B. anthracis isolates

Changes in Predominance and Diversity of Genomic Subtypes of Bordetella pertussis Isolated in the United States, 1935 to 1999

Pulsed-field gel electrophoresis (PFGE) of Bordetella pertussis chromosomal DNA fragments generated by XbaI restriction has been used to subtype isolates for epidemiologic studies. To better understand the natural history of pertussis, we determined the PFGE profiles of 1,333 strains isolated in the United States from 1935 to 1999. Results showed a shift in prevalent profiles from the earliest to the latest study periods. In addition, genetic diversity decreased over time, and prevalent profiles were more highly related to each other than to less common profiles. These results provide the foundation for investigating the impact of prevention strategies, including the use of the acellular vaccines, on the currently circulating B. pertussis population

Identification of Leptospira inadai by continuous monitoring of fluorescence during rapid cycle PCR

Seven new Leptospira isolates from rats, a buffalo, and contaminated media showed either reactive serology against more than 1 serogroup or no reactive serology against a reference panel of 22 serovars in the microscopic agglutination test (MAT). Because of these inconclusive results, the 16S rDNA sequences of these isolates were determined and found to resemble that of the type strain of Leptospira inadai (L. inadai), serovar lyme strain 10, which is considered to be nonpathogenic for humans. Comparative analyses of other Leptospira 16S rDNA sequences from databases revealed a L. inadai-specific signature sequence, against which an amplification primer was designed. This primer when used in conjunction with an universal primer enabled the trial of a rapid PCR protocol in which fluorescence emissions due to binding of SYBR Green I dye to PCR products were continuously monitored during rapid thermal cycling. A melting curve acquired immediately after PCR was used to distinguish the intended product. The thermal cycling and continuous monitoring of fluorescence emission were accomplished by the LightCycler; the whole procedure of 30 PCR cycles and melting curve acquisition required only 20 minutes. The primer achieved the required specificity, as the intended PCR product resulted only from 6 confirmed L. inadai reference strains and 7 field isolates that had been verified as L. inadai by the 16S rDNA sequencing, but not from 16 reference strains of Leptospira belonging to 7 other genospecies. Furthermore, these experiments showed that the PCR protocol was robust because target DNA of different conditions, which were extracted by either 1 of the 4 methods used, could be detected