Consecutive outbreaks of acute aflatoxicosis in Kenya in 2004 and 2005 caused > 150 deaths. In response, the Centers for Disease Control and Prevention and the World Health Organization convened a workgroup of international experts and health officials in Geneva, Switzerland, in July 2005. After discussions concerning what is known about aflatoxins, the workgroup identified gaps in current knowledge about acute and chronic human health effects of aflatoxins, surveillance and food monitoring, analytic methods, and the efficacy of intervention strategies. The workgroup also identified public health strategies that could be integrated with current agricultural approaches to resolve gaps in current knowledge and ultimately reduce morbidity and mortality associated with the consumption of aflatoxin-contaminated food in the developing world. Four issues that warrant immediate attention were identified: a) quantify the human health impacts and the burden of disease due to aflatoxin exposure; b) compile an inventory, evaluate the efficacy, and disseminate results of ongoing intervention strategies; c) develop and augment the disease surveillance, food monitoring, laboratory, and public health response capacity of affected regions; and d) develop a response protocol that can be used in the event of an outbreak of acute aflatoxicosis. This report expands on the workgroup’s discussions concerning aflatoxin in developing countries and summarizes the findings.

Objectives: During January–June 2004, an aflatoxicosis outbreak in eastern Kenya resulted in 317 cases and 125 deaths. We conducted a case–control study to identify risk factors for contamination of implicated maize and, for the first time, quantitated biomarkers associated with acute aflatoxicosis.Design: We administered questionnaires regarding maize storage and consumption and obtained maize and blood samples from participants.Participants: We recruited 40 case-patients with aflatoxicosis and 80 randomly selected controls to participate in this study.Evaluations/Measurements: We analyzed maize for total aflatoxins and serum for aflatoxin B1–lysine albumin adducts and hepatitis B surface antigen. We used regression and survival analyses to explore the relationship between aflatoxins, maize consumption, hepatitis B surface antigen, and case status.Results: Homegrown (not commercial) maize kernels from case households had higher concentrations of aflatoxins than did kernels from control households [geometric mean (GM) = 354.53 ppb vs. 44.14 ppb; p = 0.04]. Serum adduct concentrations were associated with time from jaundice to death [adjusted hazard ratio = 1.3; 95% confidence interval (CI), 1.04–1.6]. Case patients had positive hepatitis B titers [odds ratio (OR) = 9.8; 95% CI, 1.5–63.1] more often than controls. Case patients stored wet maize (OR = 3.5; 95% CI, 1.2–10.3) inside their homes (OR = 12.0; 95% CI, 1.5–95.7) rather than in granaries more often than did controls.Conclusion: Aflatoxin concentrations in maize, serum aflatoxin B1–lysine adduct concentrations, and positive hepatitis B surface antigen titers were all associated with case status.Relevance: The novel methods and risk factors described may help health officials prevent future outbreaks of aflatoxicosis.

A method for quantitative analysis of aflatoxin B1-lysine adduct (B1-Lys) in serum by liquid chromatography using tandem mass spectrometry (LC/MS/MS) is presented. The protein in a 250-microL sample was digested in the presence of a stable-isotope internal standard during a 4-h incubation at 37 degrees C with Pronasetrade mark. B1-Lys and the internal standard were extracted using mixed-mode solid-phase extraction cartridges and eluted with 2% formic acid in methanol. Following evaporation and reconstitution, extracts were injected onto a Luna C-18(2) column and eluted with a step gradient of acetonitrile and 0.06% formic acid. The B1-Lys and the internal standard were detected in a positive ionization selective reaction monitoring mode with a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer. Calibration curves were linear for concentrations from 0.05-8.0 ng/mL. The method was validated with aflatoxin B1 dosed rat serum diluted to anticipated high and low concentrations. Total imprecision determined from 30 measurements over 15 days was 5.6% and 9.1%, respectively. Recoveries of 78.8 +/- 6.4% for B1-Lys and 85.4 +/- 12.4% for the internal standard were based on the full extraction and reconstitution processes. The method can be used to quantitate B1-Lys at the 0.5 pg/mg albumin level and is suitable for routine analysis.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

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Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B 1 . In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r 2 = 0.95) and 3.3 (r 2 = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen. (Cancer Epidemiol Biomarkers Prev 2008;17(7):1653 -7)

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