Serologic surveillance of anthrax in the Serengeti ecosystem, Tanzania, 1996-2009.

Bacillus anthracis, the bacterium that causes anthrax, is responsible for varying death rates among animal species. Difficulties in case detection, hazardous or inaccessible carcasses, and misdiagnosis hinder surveillance. Using case reports and a new serologic assay that enables multispecies comparisons, we examined exposure to and illness caused by B. anthracis in different species in the Serengeti ecosystem in Tanzania during 1996-2009 and the utility of serosurveillance. High seroprevalence among carnivores suggested regular nonfatal exposure. Seropositive wildebeest and buffalo showed that infection was not invariably fatal among herbivores, whereas absence of seropositivity in zebras and frequent detection of fatal cases indicated high susceptibility. Exposure patterns in dogs reflected known patterns of endemicity and provided new information about anthrax in the ecosystem, which indicated the potential of dogs as indicator species. Serosurveillance is a valuable tool for monitoring and detecting anthrax and may shed light on mechanisms responsible for species-specific variability in exposure, susceptibility, and mortality rates.

Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization.

Quantification of anthrax lethal toxin (LTx) neutralization activity (TNA) is pivotal in assessing protective antibody responses to anthrax vaccines and for evaluation of immunotherapies for anthrax. We have adapted and redesigned the TNA assay to establish a unifying, standardized, quantitative and validated technology platform for LTx neutralization in the J774A.1 murine cell line. Critical design features of this platform are 1) the application of a free-form or constrained 4 parameter logistic (4-PL) function to model neutralization responses within and between boundary limits of 100% cell survival and 95% cell lysis and 2) to exploit innovative assay curve recognition algorithms for interpretive endpoints. The assay was validated using human serum ED50 (dilution of serum effecting 50% neutralization) as the primary reportable value (RV). Intra-operator and intermediate precision, expressed as the coefficient of variation (%CV), were high at 10.5-15.5%CV and 13.5-14.5%CV respectively. TNA assay dilutional linearity was demonstrated for human sera using linear regression analysis of log(10) transformed data with slope=0.99, intercept=-0.03 and r(2)=0.985. Assay accuracy, inferred from the precision and linearity data and using a spike-recovery approach, was high with a percent error (%E) range of only 3.4-20.5%E. The lower limit of detection (LLOD) was ED50=12 and the lower limit of quantification (LLOQ) was ED50=36. The cell-based assay was robust, tolerating incubation temperatures from 35 to 39 degrees C, CO(2) concentrations from 3% to 7% and reporter substrate (MTT) concentrations of 2.5-7.5 mg/ml. Strict assay quality control parameters were met for up to 25 cell culture passages. The long term (50 month) assay stability, determined using human reference standards AVR414 and AVR801, indicated high precision, consistent accuracy and no detectable assay drift. A customized software program provided two additional assay metrics, Quantification Titer (QT) and Threshold Titer (TT), both of which demonstrate acceptable accuracy, precision and dilutional linearity. The TT was also used to establish the assay reactivity threshold (RT). The application of the assay to sera from humans, Rhesus macaques and rabbits was demonstrated separately and by aggregate dilutional linearity analysis of the ED50 (slope=0.98, intercept=0.003, r(2)=0.989). We propose this TNA assay format with a qualified standard reference serum and customized interpretive software as a unifying platform technology for determination of functional serologic responses to anthrax vaccines and for evaluation of anthrax immunotherapeutics.

Cryptococcus gattii infection: characteristics and epidemiology of cases identified in a South African province with high HIV seroprevalence, 2002-2004.

We describe 46 Cryptococcus gattii-infected persons identified by population-based surveillance conducted in South Africa. Most patients with C. gattii infection presented with meningitis. The mortality rate during hospitalization was 36%. We found no significant differences between persons with and persons without C. gattii infection with regard to clinical presentation, acquired immunodeficiency syndrome diagnosis, concomitant conditions, or prior opportunistic infections. C. gattii isolates had low MICs to the tested antifungal drugs.

Invasive infection with multidrug-resistant Salmonella enterica serotype typhimurium definitive type 104 among HIV-infected adults.

BACKGROUND: Multidrug-resistant Salmonella enterica serotype Typhimurium definitive type 104 (MRDT104), with resistance to at least ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (R-type ACSSuT), was first detected in the United States in 1985, and the prevalence increased to account for nearly 7% of Salmonella infections in 1998. METHODS: A retrospective study of S. Typhimurium infections in an urban health care system assessed whether infection with an antibiotic-resistant strain--and specifically MRDT104--was associated with invasive disease or HIV infection. Sixty cases of S. Typhimurium infection were identified. RESULTS: Of the 50 isolates available for analysis, 30 (60%) were MRDT104. Pathogens were isolated from blood in 25 (83%) of 30 patients infected with MRDT104, compared with 10 (50%) of 20 patients who were infected with non-MRDT104 strains (P = .01). Among isolates obtained from 32 HIV-infected patients, 19 (95%) of 20 MRDT104 isolates were from blood specimens, compared with 8 (66%) of 12 non-MRDT104 isolates (P = .05). CONCLUSIONS: MRDT104 accounted for the majority of S. Typhimurium infections in this patient population, and MRDT104 infections were more invasive than non-MRDT104 infections, particularly in HIV-infected persons.

Predictability of anthrax infection in the Serengeti, Tanzania.

Anthrax is endemic throughout Africa, causing considerable livestock and wildlife losses and severe, sometimes fatal, infection in humans. Predicting the risk of infection is therefore important for public health, wildlife conservation and livestock economies. However, because of the intermittent and variable nature of anthrax outbreaks, associated environmental and climatic conditions, and diversity of species affected, the ecology of this multihost pathogen is poorly understood.We explored records of anthrax from the Serengeti ecosystem in north-west Tanzania where the disease has been documented in humans, domestic animals and a range of wildlife. Using spatial and temporal case-detection and seroprevalence data from wild and domestic animals, we investigated spatial, environmental, climatic and species-specific associations in exposure and disease.Anthrax was detected annually in numerous species, but large outbreaks were spatially localized, mostly affecting a few focal herbivores.Soil alkalinity and cumulative weather extremes were identified as useful spatial and temporal predictors of exposure and infection risk, and for triggering the onset of large outbreaks.Interacting ecological and behavioural factors, specifically functional groups and spatiotemporal overlap, helped to explain the variable patterns of infection and exposure among species.Synthesis and applications. Our results shed light on ecological drivers of anthrax infection and suggest that soil alkalinity and prolonged droughts or rains are useful predictors of disease occurrence that could guide risk-based surveillance. These insights should inform strategies for managing anthrax including prophylactic livestock vaccination, timing of public health warnings and antibiotic provision in high-risk areas. However, this research highlights the need for greater surveillance (environmental, serological and case-detection-orientated) to determine the mechanisms underlying anthrax dynamics.

Detection and quantification of anthrax lethal factor in serum by mass spectrometry.

The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.

Multidrug-resistant strains of Salmonella enterica Typhimurium, United States, 1997-1998.

To evaluate multidrug-resistant strains of Salmonella enterica serotype Typhimurium, including definitive type 104 (DT104) in the United States, we reviewed data from the National Antimicrobial Resistance Monitoring System (NARMS). In 1997 to 1998, 703 (25%) of 2,767 serotyped Salmonella isolates received at NARMS were S. Typhimurium; antimicrobial susceptibility testing and phage typing were completed for 697. Fifty-eight percent (402) were resistant to > or = 1 antimicrobial agent. Three multidrug-resistant (> or = 5 drugs) strains accounted for (74%) 296 of all resistant isolates. Ceftriaxone resistance was present in 8 (3%), and nalidixic acid resistance in 4 (1%), of these multidrug-resistant strains. By phage typing, 259 (37%) of S. Typhimurium isolates were DT104, 209 (30%) were of undefined type and 103 (15%) were untypable. Fifty percent (202) of resistant (> or = 1 drug) isolates were DT104. Multidrug-resistant S. Typhimurium isolates, particularly DT104, account for a substantial proportion of S. Typhimurium isolates; ceftriaxone resistance is exhibited by some of these strains.

Introduction; The history of public health and veterinary public service; Historic and future perspectives of the American College of Veterinary Preventive Medicine; Blue ribbon task force report on the future of Food Safety and Inspection Service veterinarians: public health professionals for the 21st century; The National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria,1996- 1999: surveillance for action; Public health education of veterinarians and veterinary students for the future; Ten great veterinary public health/preventive medicine achievements in the United States, 1901 to 2000.

Historically, veterinary medicine has made its greatest contributions in the public sector and in settings other than direct care of the individual patient. So why are so many of us unaware of our profession's achievements in this arena? My introduction to veterinary preventive medicine and public health was through military service. My obligation to serve a two-year doctor draft in the United States Air Force became an adventure for my family and me, rather than an unwelcome intrusion on my ultimate desire to return to private clinical practice. The operative words are private and clinical. Why was I unaware of the opportunities available to those of us educated in this discipline sometimes referred to as comparative medicine?

Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins.

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 microgram/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 micro g of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 microgram/ml, while the dynamic range was 0.06 to 1.7 microgram/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 microgram of anti-PA IgG per ml, the RDL was 0.016 microgram/ml, and the whole-serum equivalent MDC was 1.5 micrograms/ml. The dynamic range was 0.006 to 6.8 microgram/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r2 = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.

Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.