Tanja Popovic

Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings.

In October 2001, the first inhalational anthrax case in the United States since 1976 was identified in a media company worker in Florida. A national investigation was initiated to identify additional cases and determine possible exposures to Bacillus anthracis. Surveillance was enhanced through health-care facilities, laboratories, and other means to identify cases, which were defined as clinically compatible illness with laboratory-confirmed B. anthracis infection. From October 4 to November 20, 2001, 22 cases of anthrax (11 inhalational, 11 cutaneous) were identified; 5 of the inhalational cases were fatal. Twenty (91%) case-patients were either mail handlers or were exposed to worksites where contaminated mail was processed or received. B. anthracis isolates from four powder-containing envelopes, 17 specimens from patients, and 106 environmental samples were indistinguishable by molecular subtyping. Illness and death occurred not only at targeted worksites, but also along the path of mail and in other settings. Continued vigilance for cases is needed among health-care providers and members of the public health and law enforcement communities.

The Changing Epidemiology of Meningococcal Disease in the United States, 1992–1996

New meningococcal vaccines are undergoing clinical trials, and changes in the epidemiologic features of meningococcal disease will affect their use. Active laboratory-based, population-based US surveillance for meningococcal disease during 1992-1996 was used to project that 2400 cases of meningococcal disease occurred annually. Incidence was highest in infants; however, 32% of cases occurred in persons >/=30 years of age. Serogroup C caused 35% of cases; serogroup B, 32%; and serogroup Y, 26%. Increasing age (relative risk [RR], 1.01 per year), having an isolate obtained from blood (RR, 4.5), and serogroup C (RR, 1.6) were associated with increased case fatality. Among serogroup B isolates, the most commonly expressed serosubtype was P1.15; 68% of isolates expressed 1 of the 6 most common serosubtypes. Compared with cases occurring in previous years, recent cases are more likely to be caused by serogroup Y and to occur among older age groups. Ongoing surveillance is necessary to determine the stability of serogroup and serosubtype distribution.

Characterization of Encapsulated and Noncapsulated Haemophilus influenzae and Determination of Phylogenetic Relationships by Multilocus Sequence Typing

ABSTRACT A multilocus sequence typing (MLST) scheme has been developed for the unambiguous characterization of encapsulated and noncapsulated Haemophilus influenzae isolates. The sequences of internal fragments of seven housekeeping genes were determined for 131 isolates, comprising a diverse set of 104 serotype a, b, c, d, e, and f isolates and 27 noncapsulated isolates. Many of the encapsulated isolates had previously been characterized by multilocus enzyme electrophoresis (MLEE), and the validity of the MLST scheme was established by the very similar clustering of isolates obtained by these methods. Isolates of serotypes c, d, e, and f formed monophyletic groups on a dendrogram constructed from the differences in the allelic profiles of the isolates, whereas there were highly divergent lineages of both serotype a and b isolates. Noncapsulated isolates were distinct from encapsulated isolates and, with one exception, were within two highly divergent clusters. The relationships between the major lineages of encapsulated H. influenzae inferred from MLEE data could not be discerned on a dendrogram constructed from differences in the allelic profiles, but were apparent on a tree reconstructed from the concatenated nucleotide sequences. Recombination has not therefore completely eliminated phylogenetic signal, and in support of this, for encapsulated isolates, there was significant congruence between many of the trees reconstructed from the sequences of the seven individual loci. Congruence was less apparent for noncapsulated isolates, suggesting that the impact of recombination is greater among noncapsulated than encapsulated isolates. The H. influenzae MLST scheme is available at www.mlst.net , it allows any isolate to be compared with those in the MLST database, and (for encapsulated isolates) it assigns isolates to their phylogenetic lineage, via the Internet.

Leptospirosis in “Eco-Challenge” Athletes, Malaysian Borneo, 2000

Adventure travel is becoming more popular, increasing the likelihood of contact with unusual pathogens. We investigated an outbreak of leptospirosis in “Eco-Challenge” multisport race athletes to determine illness etiology and implement public health measures. Of 304 athletes, we contacted 189 (62%) from the United States and 26 other countries. Eighty (42%) athletes met our case definition. Twenty-nine (36%) case-patients were hospitalized; none died. Logistic regression showed swimming in the Segama River (relative risk [RR]=2.0; 95% confidence interval [CI]=1.3 to 3.1) to be an independent risk factor. Twenty-six (68%) of 38 case-patients tested positive for leptospiral antibodies. Taking doxycycline before or during the race was protective (RR=0.4, 95% CI=0.2 to 1.2) for the 20 athletes who reported using it. Increased adventure travel may lead to more frequent exposure to leptospires, and preexposure chemoprophylaxis for leptospirosis (200 mg oral doxycycline/week) may decrease illness risk. Efforts are needed to inform adventure travel participants of unique infections such as leptospirosis.

Characterization of Bacillus cereus Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to Bacillus anthracis and Harbor B. anthracis Virulence Genes

ABSTRACT Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victims worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-d-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.

Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.

Pathology and Pathogenesis of Bioterrorism-Related Inhalational Anthrax

During October and November 2001, public health authorities investigated 11 patients with inhalational anthrax related to a bioterrorism attack in the United States. Formalin-fixed samples from 8 patients were available for pathological and immunohistochemical (IHC) study using monoclonal antibodies against the Bacillus anthracis cell wall and capsule. Prominent serosanguinous pleural effusions and hemorrhagic mediastinitis were found in 5 patients who died. Pulmonary infiltrates seen on chest radiographs corresponded to intraalveolar edema and hyaline membranes. IHC assays demonstrated abundant intra- and extracellular bacilli, bacillary fragments, and granular antigen-staining in mediastinal lymph nodes, surrounding soft tissues, and pleura. IHC staining in lung, liver, spleen, and intestine was present primarily inside blood vessels and sinusoids. Grams staining of tissues was not consistently positive. In 3 surviving patients, IHC of pleural samples demonstrated abundant granular antigen-staining and rare bacilli while transbronchial biopsies showed granular antigen-staining in interstitial cells. In surviving patients, bacilli were not observed with grams stains. Pathological and IHC studies of patients who died of bioterrorism-related inhalational anthrax confirmed the route of infection. IHC was indispensable for diagnosis of surviving anthrax cases. The presence of B. anthracis antigens in the pleurae could explain the prominent and persistent hemorrhagic pleural effusions.

Mutator clones of Neisseria meningitidis in epidemic serogroup A disease

Serogroup A Neisseria meningitidis has repeatedly caused widespread epidemics of meningitis and septicemia throughout the 20th century. Recently, in a limited collection of strains, epidemic serogroup A isolates were found to have elevated mutation rates that was caused by defects in mismatch repair pathways. To ascertain the role of these mutators in the epidemic spread of this serogroup, the prevalence of hypermutability in a collection of 95 serogroup A N. meningitidis invasive isolates was determined. Overall mutability in Neisseriae can be described by measuring both missense mutation rates as well as phase variation frequencies of “contingency loci.” Fifty-seven percent of serogroup A isolates possessed elevated mutability, which could be divided into two classes: intermediate and high level. Eleven of 20 high-level mutators, with phase variation rates >100-fold higher than wild-type isolates, were defective in mismatch repair. Ten of the 34 intermediate mutators possessing >10-fold increases in phase variation rates could be partially complemented by a wild-type mutL allele. A high prevalence of mutators in epidemic isolates indicates that hypermutability may play a major role in the transmission of this pathogen. The added diversity derived from increased phase variation rates may allow fixation of mutator alleles more frequently during epidemic spread.

Fit genotypes and escape variants of subgroup III Neisseria meningitidis during three pandemics of epidemic meningitis

The genetic variability at six polymorphic loci was examined within a global collection of 502 isolates of subgroup III, serogroup A Neisseria meningitidis. Nine “genoclouds” were identified, consisting of genotypes that were isolated repeatedly plus 48 descendent genotypes that were isolated rarely. These genoclouds have caused three pandemic waves of disease since the mid-1960s, the most recent of which was imported from East Asia to Europe and Africa in the mid-1990s. Many of the genotypes are escape variants, resulting from positive selection that we attribute to herd immunity. Despite positive selection, most escape variants are less fit than their parents and are lost because of competition and bottlenecks during spread from country to country. Competition between fit genotypes results in dramatic changes in population composition over short time periods.

Evaluation and Validation of a Real-Time Polymerase Chain Reaction Assay for Rapid Identification of Bacillus anthracis

To the Editor: During the 2001 anthrax outbreak, we evaluated and validated a highly sensitive and specific three-target (two plasmid and one chromosomally located target) 5´ nuclease assay (real-time polymerase chain reaction [PCR]) for detection and identification of Bacillus anthracis. This PCR assay was successfully used to rapidly test hundreds of suspect isolates as well as screen environmental samples for the presence of B. anthracis throughout the 2001 anthrax outbreak. For the first time in an outbreak setting, a PCR assay was used to detect B. anthracis directly from clinical specimens, consequently becoming a part of the laboratory confirmation of anthrax. In this letter, we describe the evaluation of this assay on a diverse panel of bacterial isolates including isolates obtained throughout the outbreak. A supplement, which includes data on the use of this assay on environmental and clinical specimens, is online (available from http://www.cdc.gov/ncidod/EID/vol8no10/02-0393sup.htm).