A preliminary survey of mycological and fumonisin and aflatoxin contamination of African traditional herbal medicines sold in South Africa.

Traditional medicine is an important aspect of healthcare delivery in South Africa and is used by at least 70% of the country's population. The trade in medicinal plants is a multi-million rand business which is a major driver for rural economies. However, the conditions in which these plant products are transported and stored make them prone to fungal contamination which results in economic losses to the traders and pose potential health hazards to consumers. Of major concern is the possible presence of toxigenic fungi and mycotoxins. This study assessed fungal and mycotoxin contamination of African herbal products sold in Cape Town and Tshwane (formerly Pretoria) in South Africa. Of the 16 samples analyzed, 15 were contaminated with at least one of these three fungal genera: Aspergillus, Fusarium, and Penicillium. Fumonisin B(1) was present in 13 of the samples in quantities ranging from 14 to 139 microg/kg (detection limit 5 microg/kg). None of the samples was contaminated with aflatoxigenic fungi or aflatoxin (detection limit 0.5 microg/kg). This is the first study to report on mycological and mycotoxin contamination of commercial traditional African medicines in South Africa. There is a need to expand the study to other urban centers to gain enough insight into this problem and then to intervene with measures that can protect the public from potential harm.

Fusarium species and fumonisins in subsistence maize in the former Transkei region, South Africa: a multi-year study in rural villages.

Fumonisin occurrence was investigated in subsistence maize in four rural villages in each of Mbizana and Centane areas, South Africa. Samples (total 211) were analysed morphologically for Fusarium species and by high performance liquid chromatography for fumonisins. The mean incidence levels of Fusarium verticillioides in Centane good maize were 16% for both 1997 and 2000, but increased to 32% in 2003, whereas Mbizana good maize contained levels of 17% and 11% (2000 and 2003 seasons, respectively). The mean total fumonisin level in good maize in Centane for 1997 and 2000 was 575 and 975 µg/kg and 2150 µg/kg in 2003. In Mbizana, the mean total fumonisin level in good maize for 2000 was 950 µg/kg, but decreased to 610 µg/kg in 2003. The 2003 drought conditions led to a substantial increase in fumonisin levels in dry subhumid Centane, compared to humid subtropical Mbizana. This study emphasises the seasonal fluctuation in fumonisin levels.

Soil fertility factors in relation to oesophageal cancer risk areas in Transkei, southern Africa.

Soil samples were collected during certain years for the period 1982-89 from high- and low-risk areas for oesophageal cancer in Transkei, southern Africa. These samples were taken either from cultivated soils under maize monoculture, or from uncultivated soils (1989 only) adjacent to the maize fields. Analyses of mineral elements in the soil samples were performed at two independent laboratories. Furthermore, soil and maize leaf samples, from field trials in a high- and a low-risk area for oesophageal cancer were analysed. The results from this study do not agree with those reported previously for Transkei. Cultivated soils in both high- and low-risk areas were found to be highly fertile. The levels of Mn, Ni, Mg, Ca, K and soil pH were significantly higher, and Al, Fe and organic matter significantly lower in the high-risk compared with the low-risk area. Leaf analysis, although not tested statistically, indicated higher levels of Mn K, Ca and Fe, and lower levels of P, in the high-risk area.

Fumonisin contamination of a corn sample associated with the induction of hepatocarcinogenesis in rats-role of dietary deficiencies.

A corn sample associated with a field outbreak of equine leukoencephalomalacia in Pennsylvania, USA, during 1983/1984 and induced hepatotoxic and hepatocarcinogenic effects when fed to male Fischer rats was analyzed mycologically and chemically for the presence of fumonisins (FB), hydrolysed FB derivatives and aflatoxins (AFB). Fusarium verticillioides was found to be the predominant fungal contaminant in the corn sample but Aspergillus flavus was also present. Trace amounts (0.1 microg/kg) of AFB(1) and AFB(2) and a total FB level of 33.5 mg/kg (FB(1):FB(2):FB(3) ratio of 9:2.3:1) were found. No hydrolysed FB derivatives or AFG(1) and AFG(2) were detected. Based on the chemical stability of the fumonisins in different corn cultures of F. verticillioides kept at 4 degrees C over a period of 13-20 years, a level of approximately 55 mg/kg of total FB is estimated in the original corn sample. A possible role of certain dietary constituents such as the high protein content and deficiencies in certain micronutrients is evaluated to address differences in the organ-specific toxicity of FB(1) in rats using commercial, semi-purified, purified and corn-only diets.

Natural occurrence of aflatoxins in peanuts and peanut butter from Bulawayo, Zimbabwe.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that may contaminate food and pose a health risk, especially in developing countries, where there is a lack of food security and quality is subsumed by food insufficiency. Aflatoxins are the most toxic known mycotoxins and are a significant risk factor for liver and kidney cancer, teratogenicity, undernutrition, and micronutrient malabsorption in both humans and animals. The main aim of the study was to determine the extent of fungal and aflatoxin contamination in peanuts and peanut butter being sold in both the formal and informal markets in Bulawayo, Zimbabwe. Eighteen peanut samples and 11 peanut butter samples were purchased from retail shops and the informal market. Fungal contamination was determined using standard mycology culture methods, while aflatoxin contamination was determined using high-performance liquid chromatography-fluorescence detection. Four of the six peanut samples tested for fungal contamination were infected with Aspergillus flavus/parasiticus, ranging from 3 to 20% of the kernels examined, while 27% (3 of 11) of the peanut butter samples were infected with A. flavus/parasiticus. Ninety-one percent (10 of 11) of the peanut butter samples were contaminated with aflatoxins (mean, 75.66 ng/g, and range, 6.1 to 247 ng/g), and aflatoxin B1 was the most prevalent (mean, 51.0 ng/g, and range, 3.7 to 191 ng/g). Three of the 18 peanut samples were contaminated with aflatoxins (range, 6.6 to 622 ng/g). The commercial peanut butter samples had very high aflatoxin levels, and manufacturers should be sensitized to the detrimental effects of aflatoxins and measures to reduce contamination.

The mycotoxin distribution in maize milling fractions under experimental conditions.

Mycotoxin contamination of maize and maize-based food and feed products poses a health risk to humans and animals if not adequately controlled and managed. The current study investigates the effect of dry milling on the reduction of fumonisins (FB), deoxynivalenol (DON) and zearalenone (ZEA) in maize. Five composite samples, constructed to represent different mycotoxin contamination levels were degermed yielding degermed maize and the germ. The degermed maize was milled under laboratory conditions and four major milling fractions (SPECIAL, SUPER, semolina (SEM) and milling hominy feed) collected. The whole maize, degermed maize and total hominy feed (germ+milling hominy feed) were reconstructed to ensure homogenous samples for mycotoxin analyses. For comparison, commercial dry milling fractions (whole maize, SPECIAL, SUPER and total hominy feed), collected from three South African industrial mills, were analysed for the same mycotoxins and hence a more accurate assessment of the distribution between the different milling fractions. The distribution of the mycotoxins during the experimental dry milling of the degermed maize differs, with FB mainly concentrated in the SPECIAL, DON in the SEM whereas ZEA was equally distributed between the two milling fractions. Distribution of mycotoxins between the fractions obtained during commercial dry milling generally provided similar results with the total hominy feed containing the highest and the SUPER milling fractions the lowest mycotoxin levels although variations existed. Although milling is an effective way to reduce mycotoxins in maize, kernel characteristics and resultant fungal colonisation may impact on the distribution of specific mycotoxins among the different milling fractions. Differences in industrial dry milling practices and problems encountered in sampling bulk maize remain a large problem in assessing mycotoxin contamination in milling fractions intended for human consumption.

Occurrence of the C-series fumonisins in maize from the former Transkei region of South Africa.

Fumonisins are a group of structurally related mycotoxins produced mainly in maize by Fusarium verticillioides and F. proliferatum. The most abundant naturally occurring analogue is fumonisin B(1) (FB(1)), with lesser amounts of fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) occurring. The C-series fumonisins (FCs) are structurally analogous to the B-series but lack the C-1 methyl group. Good and mouldy subsistence-grown maize samples were collected from the Centane and Bizana districts in the former Transkei region of South Africa. After extraction with methanol/water and clean-up on strong anion exchange solid phase extraction cartridges, FB(1), FB(2), FB(3), FC(1), FC(3) and FC(4) were determined by reversed-phase LC-MS/MS using positive ion electrospray ionisation. FB(1) levels in both good and mouldy maize from Centane (means (±SD) 2.75 ± 2.24 and 23.4 ± 12.5 mg kg(-1), respectively) were higher than the corresponding levels in maize samples from Bizana (means 0.056 ± 0.157 and 3.71 ± 5.01 mg kg(-1), respectively). Similarly, FC(1) levels in both good and mouldy maize from Centane (means 0.107 ± 0.099 and 0.814 ± 0.391 mg kg(-1), respectively) were higher than in Bizana, where FC(1) was detected in only one (0.018 mg kg(-1)) of 19 good maize samples and occurred in mouldy maize with a mean of 0.102 ± 0.135 mg kg(-1). A significant correlation (r=0.982, p<0.01) was observed between FB(1) and FC(1) levels in all samples, with FC(1) levels at 3.3% of the corresponding FB(1) levels. FC(4) levels were similar to FC(1), whereas only low amounts of FC(3) were detected.

Individual fumonisin exposure and sphingoid base levels in rural populations consuming maize in South Africa.

Low and high oesophageal cancer incidence areas of the former Transkei region of South Africa have been associated with corresponding low and high levels of fumonisin contaminated home-grown maize. This is the first study in South Africa assessing fumonisin B (FB) mycotoxin exposure by quantifying individual maize consumption with weighed food records and FB levels from maize in each participant's household and concurrently evaluating sphinganine (Sa), sphingosine (So) and Sa/So ratios in plasma and urine of these participants as possible biomarkers of FB exposure. The high consumption of maize in Bizana (n=36) and Centane (n=30) of 0.41+/-0.21 and 0.39+/-0.19 kg/day, respectively, confirms the reliance on maize as the dietary staple. Mean total FB (FB(1)+FB(2)+FB(3)) levels in home-grown maize were 0.495+0.880 and 0.665+0.660 mg/kg in Bizana and Centane, respectively. Mean fumonisin exposure based on individual consumption was 3.9+/-7.3 and 4.1+/-7.6 microg/kg body weight/day, respectively, for Bizana and Centane. The mean combined sphinganine/sphingosine ratios in Bizana and Centane were similar and ranged from 0.10-0.55 in plasma (n=41) and urine (n=62). There was no association between sphingoid base levels and/or Sa/So ratios in the plasma and urine and individual fumonisin exposure, negating the sphingoid bases as potential biomarkers of fumonisin exposure in humans.

Simple intervention method to reduce fumonisin exposure in a subsistence maize-farming community in South Africa.

In the Centane magisterial area of South Africa, high rates of oesophageal cancer have been associated with home-grown maize contaminated with fumonisins. The aim of this study was to implement a simple intervention method to reduce fumonisin exposure in a subsistence-farming community. The hand-sorting and washing procedures, based on traditional maize-based food preparation practices, were previously customised under laboratory-controlled conditions. Home-grown maize and maize-based porridge collected at baseline were analysed for fumonisin B(1), B(2) and B(3). The geometric mean (95% confidence interval) of fumonisin contamination in the home-grown maize at baseline was 1.67 (1.21-2.32) mg kg(-1) and 1.24 (0.75-2.04) mg kg(-1) (dry weight) in the porridge. Fumonisin exposure was based on individual stiff porridge consumption and the specific fumonisin levels in the porridge (dry weight) consumed. Porridge (dry weight) consumption at baseline was 0.34 kg day(-1) and fumonisin exposure was 6.73 (3.90-11.6) µg kg(-1) body weight day(-1). Female participants (n = 22) were trained to recognise and remove visibly infected/damaged kernels and to wash the remaining maize kernels. The discarded kernels represented 3.9% by weight and the fumonisins varied from 17.1 to 76.9 mg kg(-1). The customised hand-sorting and washing procedures reduced fumonisin contamination in the maize and porridge by 84 and 65%, respectively. The intervention reduced fumonisin exposure by 62% to 2.55 (1.94-3.35) µg kg(-1) body weight day(-1). This simple intervention method has the potential to improve food safety and health in subsistence-farming communities consuming fumonisin-contaminated maize as their staple diet.

Sphingoid base levels in humans consuming fumonisin-contaminated maize in rural areas of the former Transkei, South Africa: a cross-sectional study.

High incidences of oesophageal cancer are associated with the consumption of subsistence-grown maize by rural populations in the former Transkei region of Eastern Cape Province, South Africa. This cross-sectional study was conducted in the north-eastern magisterial area of Bizana (a previously low oesophageal cancer incidence area) and the south-eastern area of Centane (a previously high incidence area). Plasma and urine samples of male and female participants were analysed for the sphingoid bases, sphinganine and sphingosine. Good home-grown and visibly mouldy maize samples, collected from the households of the participants, were analysed for fumonisin B(1), B(2) and B(3). Plasma sphinganine/sphingosine ratios in males and females were significantly lower (p < 0.05) due to lower sphinganine levels in Bizana compared to Centane. In contrast, the urinary female and combined (males + females) sphinganine/sphingosine ratios were significantly higher (p < 0.05) in Bizana due to the significantly lower (p < 0.05) urinary sphingosine levels. Interestingly, urinary sphingoid base levels were significantly lower (p < 0.05) in males than females within each area. Based on the mean total fumonisin levels in good maize, the estimated mean probable daily intake (PDI) was 5.8 microg kg(-1) body weight day(-1) in Bizana during 2000 and 4.4 and 6.7 5.8 microg kg(-1) body weight day(-1) in Centane during 1997 and 2000, respectively, exceeding the maximum tolerable daily intake proposed by JECFA. However, there was no significant difference in the mean total fumonisin levels in the maize between the magisterial areas. The observed differences in plasma and urinary sphingoid base levels could not be ascribed as a biomarker of fumonisin exposure and further studies at an individual level are required.

Exposure assessment for fumonisins in the former Transkei region of South Africa.

The fumonisins are mycotoxins produced mainly by Fusarium verticillioides and F. proliferatum in maize, the predominant cereal staple for subsistence farming communities in southern Africa. In order to assess exposure to these mycotoxins in the Bizana (now known as Mbizana) and Centane magisterial areas of the former Transkei region of the Eastern Cape Province of South Africa, the actual maize consumption by different age groups in these communities was measured. In the groups 1-9 years (n = 215) and 10-17 (n = 240) years, mean consumption (+/-standard error) was 246 +/- 10.8 and 368 +/- 10.3 g per person day(-1), respectively, with no significant difference (p > 0.05) between the magisterial areas. For adults (18-65 years) mean maize consumption in Bizana (n = 229) and Centane (n = 178) were significantly different (p < 0.05) at 379 +/- 10.5 and 456 +/- 11.9 g per person day(-1), respectively. An exposure assessment was performed by combining the maize consumption distribution with previously determined levels of total fumonisin (fumonisins B(1) and B(2) combined) contamination in home-grown maize in these two areas. Assuming an individual adult body weight of 60 kg, fumonisin exposure in Bizana, an area of relatively low oesophageal cancer incidence, was 3.43 +/- 0.15 microg kg(-1) body weight day(-1), which was significantly lower (p < 0.05) than that in Centane (8.67 +/- 0.18 microg kg(-1) body weight day(-1)), an area of high oesophageal cancer incidence. Mean fumonisin exposures in all age groups in both Bizana and Centane were above the provisional maximum tolerable daily intake (PMTDI) of 2 microg kg(-1) body weight day(-1) set by the Joint FAO/WHO Expert Committee on Food Additives.

Fusarium species from plant debris associated with soils from maize production areas in the Transkei region of South Africa.

Fusarium species were isolated from plant debris in soil samples collected from cultivated maize fields and from undisturbed grasslands in two areas of the Transkei region. A total of 1205 Fusarium isolates were recovered from 27 soil samples. Fifteen Fusarium species were recovered from plant debris from Bizana soils and 13 Fusarium species from plant debris from Centane soils. The two dominant Fusarium species in both areas were F. oxysporum and F. equiseti. Very few isolates of F. moniliforme and F. subglutinans were recovered, but both of these species had significantly higher relative densities in cultivated soils than in undisturbed soils.

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures.

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.

Evalution of liquid media for fumonisin production byFusarium moniliforme MCR 826.

Production of fumonlsins B1 (FB1) and B2 (FB2) by 5 lyophillzation batches ofFusarium moniliforme strain MRC 826 was studied in several liquid media and vermlculite supplemented with liquid media. In addition the effect of different parameters including pH, Inert material, shake versus stationary cultures as well as different carbon sources on the production of the fumonlsins were investigated. Fumonlsin production in liquid cultures was significantly (P<0.01) correlated (r=0.92-0.98) with fungal growth, which in turn is affected by the pH of the medium as well as the carbon source utilized. The highest FB1 yields (approximately 40 mg/l) over the incubation period of 14 days were produced in a chemically defined medium with glucose as carbon source set at an initial pH value of 4. FB1 production in "corn patty" cultures (approximately 1 to 3 g/kg), however, by far exceeded that obtained in the liquid media, while poor fungal growth and fumonlsin production was obtained in vermlculite supplemented cultures. From these studies it became clear that the ability of a culture to produce fumonlsins is determined by the interaction of a variety of physiological and nutritional factors regarding the inoculum and the culture medium.

Fusarium dimerum as a cause of human eye infections.

Fusarium dimerum, typically a soil fungus, was isolated from an adult male suffering from a corneal ulcer following an injury to the eye. This fungus has not been described to cause human infections in South Africa and has not been recorded from soil, plant or organic material in this country. The macro- and microscopic characteristics of the isolate were found to be indistinguishable from described strains. Its authenticity was confirmed by comparing it to other human isolates from the eye obtained in the USA, thus rendering this the first report of F. dimerum from an eye infection in a human in South Africa.

The influence of deck storage and initial processing on patulin levels in apple juice.

Patulin, a secondary metabolite produced by Penicillium expansum and some other fungal species, is a common contaminant of ripened apples used for the production of apple juice concentrates. The limited availability of suitable storage facilities may result in fruit being subjected to storage in the open ('deck storage') for extended periods of time, prior to processing. A study was conducted to determine the influence that deck storage and subsequent initial processing practices had on patulin levels in freshly pressed juice. Over the study period, triplicate samples were collected at four strategic processing points from individual consignments of Granny Smith apples deck-stored for 7, 15 and 33 days, respectively. Over the study period, mean patulin levels in non-processed fruit increased from 90 to 2445 ng/g, respectively, but decreased to between 75 and 695 ng/g, respectively, following a water wash step. Subsequent removal of rotten/damaged fruit decreased patulin levels further (to between 55 and 405 ng/g, respectively), although the numerical decreases between sampling points were not shown to be statistically significant (P > 0.05). However, patulin levels were significantly higher (P < 0.05) in the rejected rotten/damaged fruit (mean levels ranged from 1120 to 6235 ng/g, respectively). P. expansum was the major patulin-producing fungus isolated from the juice samples. The mycological analyses tended to support the chemical data, in that removal of the rotten/damaged fractions significantly reduced total fungal counts in the juice samples.

Ear-rot fungi and mycotoxins in South African corn of the 1989 crop exported to Taiwan.

A shipment of South African corn (1989) exported to Taiwan, was analyzed for various ear-rot fungi and Fusarium mycotoxins. Two sets of samples, one from the points of origin in South Africa prior to shipment, and the other from the end-point distributors in Taiwan, were studied. Surface-sterilized kernels were plated onto two different agar media and the fungal colonies identified. High Performance Liquid Chromatography was used to analyze mycotoxin levels. The predominant ear-rot fungi, in decreasing order of isolation frequency, were Fusarium subglutinans, F. moniliforme, Diploidia maydis and F. graminearum. Aspergillus flavus and A. parasiticus were not isolated from samples prior to export, but a small number of A. flavus isolates were found after shipment. The predominant mycotoxins were fumonisins B1 (0-865 ng/g) and B2 (0-250 ng/g). Low levels of moniliformin (< or = 390 ng/g) were detected in some samples before shipment. Zearalenone (25 ng/g), and nivalenol (120 ng/g) were detected in two out of 32 samples taken in Taiwan. The samples contained no detectable levels of either aflatoxins (> 0.5 ng/g) or deoxynivalenol (> 100 ng/g) before or after shipment.