Molecular Characterization of Anthrax in Positive Powders: a Mexican Experience

Recently we read contributions from La Scola et al. (4), Luna et al. (5), and Bell et al. (1) published in this journal about their experiences with screening and laboratory analysis carried out to identify anthrax in human samples (nasal swabs) and environmental samples (mail pieces and packages), essentially by using B. anthracis DNA detection by LightCycler PCR. This paper reports our experience with extensive screening of suspected mail and packages with anthrax spores using biochemical testing of isolates and conventional PCR techniques.

Proximity to the United States, which is a main target for a bioterrorist attack, and intense trading of merchandise demanded a response of the Mexican public health system. It established several strategies, one of which was to reinforce laboratory surveillance for anthrax.

Suspicious pieces were obtained from the Mexican Postal Service or the population and sent to the nearest public health laboratory for presumptive identification; those positive samples were sent to the national laboratory for confirmation. If the packages did not contain any dust, a scraping of the internal surface with a sterilized swab was taken. If a package contained dust, a sample was taken, and in both cases the sample was put into 5 ml of distilled sterile water with 5 ml of tripticase medium and incubated at 62.5°C for 15 min to induce spore germination. Subsequently, several dilutions were placed in sheep blood and polymyxin-lysosyme-EDTA-thallous acetate selective agars. After incubation for 24 h at 37°C, colonies developed were Gram stained, and those with suggestive morphology were tested with the API CHB 50 biochemical assay (Biomerieux, Anglum Road, Mo.). Simultaneously, testing of motility, penicillin susceptibility, and phage gamma lysis (donated by the Centers for Disease Control) was done. To confirm bacterial identity, nested PCR was carried out to detect the virulence determinants edema factor (EF) in pXO1 and Cap C genes in the plasmid pXO2 (2, 3). Colonies of B. anthracis, Sterne and Pasteur strains, were used as positive controls, and Bacillus cereus (ATTC 14579), Bacillus subtilis, Escherichia coli and Streptococcus faecalis were used as negative controls.

We analyzed 6,230 suspicious pieces. All nonhemolytic colonies detected were processed as mentioned above. Only 37 isolates were strongly suggestive and were analyzed by nested PCR, and four were positive for the B. anthracis Sterne vaccine strain. The virulence associated with strains of B. anthracis is due to exotoxin compounded by protective antigen, EF, and lethal factor, which are placed in pXO1, and by poly-d-glutamic acid capsular polypeptide (located in pXO2). In our four positive samples we detected only pXO1. This demonstrated that all mail pieces analyzed did not contain the pathogen B. anthracis and these four isolates correspond to vaccine strains, since live vaccines are developed using strains that contain only pXO1 and which do not produce the capsule proteins. Regarding efficiency and efficacy, we concluded that using (i) several screening steps and (ii) a conventional PCR, the laboratory network was able to give a B. anthracis-specific characterization; nevertheless, real-time PCR is currently preferred.