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 Microorganisms Isolated From Counterfeit and Unapproved Decorative Contact Lenses.

All contact lenses (corrective/noncorrective) are considered Class II or Class III medical devices under the Federal Food, Drug, and Cosmetic Act, which also states that contact lenses can only be obtained with a prescription. The Forensic Chemistry Center of the US Food & Drug Administration has examined over 300 decorative, noncorrective contact lenses obtained without a prescription. Our observations indicate that 60% of the counterfeit lenses and 27% of the unapproved lenses examined were positive for microbial contamination. Twenty-nine different brands of noncorrective contact lenses were examined, and 48% of them had at least one sample positive for microbial contamination. Each microorganism was further identified using DNA sequencing. Contaminated contact lenses are associated with numerous health risks, including ocular infections and conjunctivitis leading to permanent visual impairment or blindness. These results support the contention that acquiring contact lenses without a prescription is a considerable threat to consumer health and safety.

Detection of sesame seed DNA in foods using real-time PCR.

The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.

Evaluation of an in vitro bioassay for the detection of purified ricin and castor bean in beverages and liquid food matrices.

The potential use of ricin as a biological weapon in food highlights the necessity for the development of food-specific detection methods. Current methods for the detection of ricin consist of various immunoassays, which detect only one subunit of the ricin toxin and therefore may not be indicative of a biologically active molecule. An in vivo assay, such as a mouse bioassay, can indicate the biological activity of the toxin; however, this method is not feasible for laboratories that do not have animal testing facilities. The purpose of this study was to develop an in vitro assay for the detection of biologically active ricin in beverages and liquid foods. Acidic and high-protein beverages were spiked with either purified ricin or ground castor beans and added to cultured human Jurkat cells. After an overnight incubation, the supernatant was tested for lactate dehydrogenase (LDH) activity with a colorimetric assay. LDH was released from the cytosol upon cell damage and was positively correlated with cell death. Ricin was detectable in all the matrices tested, with a sensitivity of 10 to 100 pg/ml. Biologically active ricin was detectable in all the matrices incubated with ground castor bean material. This method provides a confirmatory way to detect biologically active ricin that can be utilized by laboratories lacking animal facilities.

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.

Detection of cashew nut DNA in spiked baked goods using a real-time polymerase chain reaction method.

The detection of potentially allergenic foods, such as tree nuts, in food products is a major concern for the food processing industry. A real-time polymerase chain reaction (PCR) method was designed to determine the presence of cashew DNA in food products. The PCR amplifies a 67 bp fragment of the cashew 2S albumin gene, which is detected with a cashew-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other tree nut species, such as almond, Brazil nut, hazelnut, and walnut, as well as 4 varieties of peanut. This assay was sensitive enough to detect 5 pg purified cashew DNA as well as cashew DNA in a spiked chocolate cookie sample containing 0.01% (100 mg/kg) cashew.

Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

Detection of crustacean DNA and species identification using a PCR-restriction fragment length polymorphism method.

The detection of potentially allergenic proteins, such as those derived from crustaceans, in food products is a major concern for the food processing industry. A PCR-restriction fragment length polymorphism (PCR-RFLP) method was designed to detect the presence of crustacean DNA in food products and to determine the species source of the DNA. This PCR assay amplifies an approximately 205-bp fragment of the 16S rRNA gene in crustacean species, including shrimp, crab, lobster, and crawfish. This reaction will not amplify DNA derived from mammals, such as cow and sheep. After amplification, the PCR product is digested with differential restriction endonucleases to determine the species source of the crustacean DNA. The specificity of this assay was demonstrated using four species of shrimp, three species of crab, and two species of lobster and crawfish. This assay is sensitive enough to detect crustacean DNA in a raw meat mixture containing <0.1% shrimp.

Characterization of microorganisms isolated from counterfeit toothpaste.

The appearance of potentially counterfeit "Colgate" toothpaste on the American market prompted a criminal investigation by the United States Food and Drug Administration (FDA), including the collection of c. 60,000 tubes of toothpaste from retail outlets and product distributors. Microbiological testing was performed based on the FDA Bacteriological Analytical Manual, which determined the presence and number of bacteria present in the products. Bacteria were isolated from each "Colgate" variety; up to 2 × 10(6) cfu/g were isolated from some of the product units. Using conventional microscopic and biochemical bacterial identification methods, most of the bacteria isolated from these samples were Gram-negative rods of several genera, including Pseudomonas, Serratia, and Klebsiella. Most of the organisms isolated represent opportunistic pathogens, and therefore, counterfeit "Colgate" toothpaste containing high levels of bacteria pose a human health hazard.