Description of a Mass Poisoning in a Rural District in Mozambique: The First Documented Bongkrekic Acid Poisoning in Africa.

Background: On 9 January 2015, in a rural town in Mozambique, >230 persons became sick and 75 died of an illness linked to drinking pombe, a traditional alcoholic beverage. Methods: An investigation was conducted to identify case patients and determine the cause of the outbreak. A case patient was defined as any resident of Chitima who developed any new or unexplained neurologic, gastrointestinal, or cardiovascular symptom from 9 January at 6:00 am through 12 January at 11:59 pm. We conducted medical record reviews, healthcare worker and community surveys, anthropologic and toxicologic investigations of local medicinal plants and commercial pesticides, and laboratory testing of the suspect and control pombe. Results: We identified 234 case patients; 75 (32%) died and 159 recovered. Overall, 61% of case patients were female (n = 142), and ages ranged from 1 to 87 years (median, 30 years). Signs and symptoms included abdominal pain, diarrhea, vomiting, and generalized malaise. Death was preceded by psychomotor agitation and abnormal posturing. The median interval from pombe consumption to symptom onset was 16 hours. Toxic levels of bongkrekic acid (BA) were detected in the suspect pombe but not the control pombe. Burkholderia gladioli pathovar cocovenenans, the bacteria that produces BA, was detected in the flour used to make the pombe. Conclusions: We report for the first time an outbreak of a highly lethal illness linked to BA, a deadly food-borne toxin in Africa. Given that no previous outbreaks have been recognized outside Asia, our investigation suggests that BA might be an unrecognized cause of toxic outbreaks globally.

Identification of the potent toxin bongkrekic acid in a traditional African beverage linked to a fatal outbreak.

In January 2015, 75 people died and 177 were hospitalized in the Mozambique village of Chitima after attending a funeral. The deaths were linked to the consumption of a traditional African beverage called pombe. Samples of the suspect pombe were subjected to myriad analyses and compared to a control sample. Ultimately, non-targeted liquid chromatography-mass spectrometry screening revealed the presence of the potent toxin bongkrekic acid, and its structural isomer, isobongkrekic acid. Quantitative analysis found potentially fatal levels of these toxins in the suspect pombe samples. Bongkrekic acid is known to be produced by the bacterium Burkholderia gladioli pv. cocovenenans. This bacterium could not be isolated from the suspect pombe, but bacteria identified as B. gladioli were isolated from corn flour, a starting ingredient in the production of pombe, obtained from the brewer's home. When the bacteria were co-plated with the fungus Rhizopus oryzae, which was also isolated from the corn flour, synergistic production of bongkrekic acid was observed. The results suggest a mechanism for bongkrekic acid intoxication, a phenomenon previously thought to be restricted to specific regions of Indonesia and China.

Improvements in the methodology of monitoring sulfur mustard exposure by gas chromatography-mass spectrometry analysis of cleaved and derivatized blood protein adducts.

An analytical method for determining exposure to 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD) has been enhanced. The method is based on the cleavage of adducted HD (protein-hydroxyethylthioethyl esters) to produce thiodiglycol. Following cleavage, a deuterated internal standard is added, and the analytes are extracted, derivatized, and analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry. Inclusion of a concentration step, addition of solid sodium bicarbonate to neutralize excess derivatization reagent, and optimization of method and instrument conditions provided dramatic increases in signal-to-noise ratio. A five-day precision and accuracy study was conducted, including interday and intraday unknown analysis. Linearity was verified by a R(2) > 0.9995 for all five curves evaluated. The precision and accuracy of the assay were demonstrated to be excellent by evaluation of the interday and intraday unknown samples (< 10% relative standard deviation and relative error in most cases). Statistical treatment of the method blanks and calibration results demonstrated a reduction in the limit of quantitation from 25 nM (HD, human plasma, in vitro) to 1.56 nM. Sample and calibration stability through the analytical sequence was established by the inclusion of continuing calibration verification standards (< 5% error). Short-term sample stability was verified by reinjection of a calibration set after 18 days (R(2) = 0.9997). Quantitative agreement with the previous method was supported by the analysis of a 50 nM standard protein sample (HD, rat plasma) with both methodologies (< 1% error).

Gas chromatographic-mass spectrometric analysis of sulfur mustard-plasma protein adducts: validation and use in a rat inhalation model.

Sulfur mustard (HD) is an alkylating agent that reacts rapidly with macromolecular targets resulting in the formation of stable adducts providing depots for markers of exposure. The purpose of this study was to validate an analytical procedure for detection of HD-plasma protein adducts and to establish the utility of the method in an HD rat inhalation study. Calibration curves were prepared in human and rat plasma at six levels of HD (12.5 to 400 nM). Correlation coefficients for the mean data were 0.9987 for human and 0.9992 for rat plasma. The percent coefficient of variation (%CV) derived from the mean concentration data ranged from 0.53 to 14.1% in human (n = 5) and 0.57 to 10.63% in rat (n = 6) plasma. Intraday and interday precision and accuracy studies were conducted at three concentration levels (25, 150, 300 nM) to represent low, medium, and high concentrations of HD relative to those employed in the calibration curve. Precision and accuracy were assessed by determining %CV and % error, respectively. For intra- and interday studies, the %CVs and absolute % errors were less than 15%. The limits of quantitation were 20.88 nM for human and 16.73 nM for rat plasma. In animal studies, rats received nebulized HD at six doses. The data indicate a dose-dependent relationship between maximal plasma concentrations and dose administered (R(2) = 0.9728). Results from this study indicate an accurate, precise, and sensitive method. The method was useful in determining plasma protein adduct formation in a rat inhalation model.