Annals of Tropical Paediatrics International Child Health
ISSN: 0272-4936 (Print) 1465-3281 (Online) Journal homepage: http://www.tandfonline.com/loi/ypch19
Aflatoxins and kwashiorkor in Durban, South Africa G. Ramjee, P. Berjak, M. Adhikari & M. F. Dutton To cite this article: G. Ramjee, P. Berjak, M. Adhikari & M. F. Dutton (1992) Aflatoxins and kwashiorkor in Durban, South Africa, Annals of Tropical Paediatrics, 12:3, 241-247, DOI: 10.1080/02724936.1992.11747579 To link to this article: http://dx.doi.org/10.1080/02724936.1992.11747579
Published online: 13 Jul 2016.
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Citing articles: 18 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypch19 Download by: [Australian Catholic University]
Date: 26 July 2017, At: 22:04
Annals of Tropical Paediatrics (1992) 12, 241-247
Aftatoxins and kwashiorkor in Durban, South Africa G. RAMJEE*t, P. BERJAKt, M. ADHIKARI* & M. F. DUTTON** Departments of* Paediatrics and Child Health and t Biology, University of Natal, Durban and Department of**Biochemistry, University of Natal, Pietermaritzburg, South Africa (Received 3 October 1991)
Summary The present investigation has indicated that maize seeds stored under various simulated seasonal conditions show a spectrum of fungi that appear as a succession. The aflatoxin-producing fungus, Aspergil/usfiavus, is favoured by storage conditions of high temperature and humidity (summer and autumn seasons). This coincides with the more frequent admission of children suffering from kwashiorkor at King Edward VIII Hospital in Durban. Aflatoxin analysis was undertaken on 74 children diagnosed at King Edward VIII Hospital in Durban as cases of kwashiorkor, marasmus or underweight (Wellcome classification). The control group consisted of 35 age-matched patients with no symptoms of protein energy malnutrition. Aflatoxins were detected in serum and/or urine from all groups, including the controls. The serum/urine ratio was significantly higher in the kwashiorkor group than in the other groups. The control group, however, had a higher proportion of urine aflatoxins than the kwashiorkor group. These findings were interpreted in terms of impaired liver function in kwashiorkor. Aflatoxins may have a role in the pathogenesis of kwashiorkor, although the present findings do not indicate that they are a causal factor.
Introduction Malnutrition is widespread and is one of the most important factors contributory to the high incidence of illness and death in developing countries. Protein energy malnutrition (PEM), which includes the conditions of kwashiorkor, marasmus and marasmic kwashiorkor, embraces all disorders attributable to deficiency of protein in the diet. 1 Kwashiorkor was first described in the 1930s. 2 However, the precise aetiology of the disease still remains obscure. Recent reports have suggested that aflatoxins, which are mycotoxins produced by the fungi Aspergillus ftavus and Aspergillus parasiticus, may Reprint requests to: Mrs Gita Ramjee, Department of Paediatrics and Child Health, University of Natal, PO Box 17039, Congella 4013, Durban, South Mrica.
have a role in the pathogenesis of kwashiorkor. 3--5 Aflatoxins are primarily metabolized in the liver by the microsomal mixed function oxidase system and bind covalently to nucleic acids and proteins, which is probably the basis of their toxic and carcinogenic effects. 6 The Aspergillus ftavus spp. are reported to contaminate many food commodities in tropical climates. 7- 9 Maize is the dietary staple of most of the black population in South Africa, including the greater Durban area, which has a high relative humidity and temperature during summer and autumn. Such conditions are conducive to rapid fungal contamination and/or proliferation of fungal contaminants already present in poorly stored feed and foodstuffs. It is during these seasons that children suffering from kwashiorkor are seen most frequently at King Edward VIII Hospital in Durban. The
242
G. Ramjee et al.
TABLE I. Storage of maize seeds under various simulated seasonal conditions Simulated seasonal conditions Summer Autumn Natal Winter Transvaal Winter
Relative humidity
Day temp.
Night temp.
(%)
CCC)
CCC)
90 90 90 65
25 25
25
18 18
18 8 8
children suffering from kwashiorkor usually come from rural areas and from low income families. The present study was initiated because it had been found that autopsy liver samples from children in Durban suffering from kwashiorkor showed the presence of aflatoxins.10 This study was aimed at ascertaining whether PEM in children could be correlated with evidence of aflatoxin consumption. In addition, a pilot study was undertaken to examine the local seed storage mycoflora (A. fiavus in particular) under various simulated seasonal conditions of storage, particularly in the Natal province.
Africa. Having obtained ethical and parental consent for the study, patients were classified as cases of kwashiorkor, marasmus or underweight (Wellcome classification). Well nourished age-matched children who had arrived at the hospital and were being investigated for other conditions were taken as controls. For every one or two malnourished patients a control child of the same age was selected. Altogether, 109 consecutive patients were admitted to the study. Blood for aflatoxin analysis was taken in the Outpatients Department on the day of admission. Collection of a urine sample, however, was not always possible. All samples were kept frozen prior to analysis. Aflatoxin analysis was carried out by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) using fluorescence detection." Analysis of serum was performed after hexane partitioning, chloroform extraction and silica-column clean-up. Detection of major aflatoxins Bp B 2, G" G 2 and M 1 was facilitated using the above methodology. The lower limits of detection for aflatoxins (pg/ml) were as follows: serum: B" 25; B2 , 1; Gp 50; G 2 , 1; M" 5; urine: B" 25; B2 , 10; G" 25; G 2, 10; Mp 5.
Methods Seed storage Mycofiora pilot study
Newly harvested maize seeds were stored in sterilized, sealed containers under various conditions of temperature and relative humidity (RH) to simulate the seasons of autumn and summer in Natal, Transvaal winter (TW) and Natal winter (NW) (Table I). Seeds were tested for fungal contamination and moisture content (MC) at the outset and after 3, 6, 9 and 12 weeks of storage.
Patients Children between the ages of 6 months and 2 years with malnutrition were selected from the Paediatric Outpatient Department of King Edward VIII Hospital, Durban, South
Statistical analysis Chi-square was used with Yates' correction. If an expected cell value was < 5 then Fisher's Exact test was used.
Results Seed-storage mycofiora (Figs. 1a, 1b, 2a, 2b)
Fungal contamination prior to storage (0 time) showed that a mixed mycoflora comprising Fusarium spp. ( 100%) and Penicillium spp. (94%) dominated the seeds' internal environment. Proliferation of Aspergillus spp. generally was evident throughout the storage period. However, under summer and autumn conditions (Figs. 1a, 1b) proliferation of A. fiavus
Aflatoxins and kwashiorkor in South Africa
"0 Q)
ti Q) ~
120
I
D
Fusarium spp.
Other Aspergilli
~ A. ochraceus
Penicillium spp.
-
Moisture content
A. flavus gp. spp
243
A.
"' "' af!. "0 Q) Q)
"0 Q)
ti Q)
#
~
(/)
CD CD
"' "' af!.
c.
"0 Q) Q)
~ ....
0
0
3
6
9
12
Time in weeks
FIG. 1. Storage of maize seeds. A. Summer, B. Autumn.
and A. parasiticus (presently considered together as both are group species of A. flavus), which was negligible after 3 and 6 weeks of storage, peaked at 100% at 9 weeks, declining to an insignificant degree thereafter. These storage conditions were: 25°C and 90% RH, and alternating between 25°C by day and 18°C at night, also at 90% RH, respectively. However, storage under generally cooler conditions (NW [Fig. 2a]) and (TW [Fig. 2b]) was correlated with a low but persistent (especially under the latter conditions) incidence of A.flavus spp., although moisture content was relatively high. Under NW conditions, seed moisture was not consistently at a high level, permitting A.flavus spp. to proliferate, 12 which might explain its more erratic isolation compared with that of seeds from the TW regimes. Other fungal species of A. ochraceus, A. glaucus and A. tirreus (classified as 'other Aspergilli'), Penicillium spp. and Fusarium spp. were also predominant under all storage conditions (Figs. 1a, band 2a, b).
Results Serum analysis Serum analysis for aflatoxin was carried out on controls (35), marasmic ( 13), underweight (16) and kwashiorkor (45) children ranging in age from 6 months to 2 years. Aflatoxins were detected in the serum of 56% of both kwashiorkor and underweight cases, 31% of those with marasmus, and 49% of control infants. There were no significant differences among the various groups in the percentage of aflatoxin-positive results (Table II).
Individual serum aflatoxins Prevalence. Serum from the kwashiorkor group showed a greater prevalence of the individual aflatoxins than that from the other groups, aflatoxin B1 (18%), aflatoxin B2 (27%) and aflatoxin G 2 (20%) being detected more frequently. There was a consistent presence of aflatoxin M 1 in all groups except the marasmic. The mean percentage
244
G. Ramjee et al.
Ill A. flavus gp. spp -g
120
t>
~ Other Aspergilli
[ ] Fusarium spp. ~ A. ochraceus
I§!S Penicillium spp.
-
A. 30
Moisture content
Q)
1; rn
20
80
3:::
"0 Q) Q)
rn Q) ~ rn
"0
"'
80
3:::
Q) Q)
0
rn
ISSN: 0272-4936 (Print) 1465-3281 (Online) Journal homepage: http://www.tandfonline.com/loi/ypch19
Aflatoxins and kwashiorkor in Durban, South Africa G. Ramjee, P. Berjak, M. Adhikari & M. F. Dutton To cite this article: G. Ramjee, P. Berjak, M. Adhikari & M. F. Dutton (1992) Aflatoxins and kwashiorkor in Durban, South Africa, Annals of Tropical Paediatrics, 12:3, 241-247, DOI: 10.1080/02724936.1992.11747579 To link to this article: http://dx.doi.org/10.1080/02724936.1992.11747579
Published online: 13 Jul 2016.
Submit your article to this journal
View related articles
Citing articles: 18 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypch19 Download by: [Australian Catholic University]
Date: 26 July 2017, At: 22:04
Annals of Tropical Paediatrics (1992) 12, 241-247
Aftatoxins and kwashiorkor in Durban, South Africa G. RAMJEE*t, P. BERJAKt, M. ADHIKARI* & M. F. DUTTON** Departments of* Paediatrics and Child Health and t Biology, University of Natal, Durban and Department of**Biochemistry, University of Natal, Pietermaritzburg, South Africa (Received 3 October 1991)
Summary The present investigation has indicated that maize seeds stored under various simulated seasonal conditions show a spectrum of fungi that appear as a succession. The aflatoxin-producing fungus, Aspergil/usfiavus, is favoured by storage conditions of high temperature and humidity (summer and autumn seasons). This coincides with the more frequent admission of children suffering from kwashiorkor at King Edward VIII Hospital in Durban. Aflatoxin analysis was undertaken on 74 children diagnosed at King Edward VIII Hospital in Durban as cases of kwashiorkor, marasmus or underweight (Wellcome classification). The control group consisted of 35 age-matched patients with no symptoms of protein energy malnutrition. Aflatoxins were detected in serum and/or urine from all groups, including the controls. The serum/urine ratio was significantly higher in the kwashiorkor group than in the other groups. The control group, however, had a higher proportion of urine aflatoxins than the kwashiorkor group. These findings were interpreted in terms of impaired liver function in kwashiorkor. Aflatoxins may have a role in the pathogenesis of kwashiorkor, although the present findings do not indicate that they are a causal factor.
Introduction Malnutrition is widespread and is one of the most important factors contributory to the high incidence of illness and death in developing countries. Protein energy malnutrition (PEM), which includes the conditions of kwashiorkor, marasmus and marasmic kwashiorkor, embraces all disorders attributable to deficiency of protein in the diet. 1 Kwashiorkor was first described in the 1930s. 2 However, the precise aetiology of the disease still remains obscure. Recent reports have suggested that aflatoxins, which are mycotoxins produced by the fungi Aspergillus ftavus and Aspergillus parasiticus, may Reprint requests to: Mrs Gita Ramjee, Department of Paediatrics and Child Health, University of Natal, PO Box 17039, Congella 4013, Durban, South Mrica.
have a role in the pathogenesis of kwashiorkor. 3--5 Aflatoxins are primarily metabolized in the liver by the microsomal mixed function oxidase system and bind covalently to nucleic acids and proteins, which is probably the basis of their toxic and carcinogenic effects. 6 The Aspergillus ftavus spp. are reported to contaminate many food commodities in tropical climates. 7- 9 Maize is the dietary staple of most of the black population in South Africa, including the greater Durban area, which has a high relative humidity and temperature during summer and autumn. Such conditions are conducive to rapid fungal contamination and/or proliferation of fungal contaminants already present in poorly stored feed and foodstuffs. It is during these seasons that children suffering from kwashiorkor are seen most frequently at King Edward VIII Hospital in Durban. The
242
G. Ramjee et al.
TABLE I. Storage of maize seeds under various simulated seasonal conditions Simulated seasonal conditions Summer Autumn Natal Winter Transvaal Winter
Relative humidity
Day temp.
Night temp.
(%)
CCC)
CCC)
90 90 90 65
25 25
25
18 18
18 8 8
children suffering from kwashiorkor usually come from rural areas and from low income families. The present study was initiated because it had been found that autopsy liver samples from children in Durban suffering from kwashiorkor showed the presence of aflatoxins.10 This study was aimed at ascertaining whether PEM in children could be correlated with evidence of aflatoxin consumption. In addition, a pilot study was undertaken to examine the local seed storage mycoflora (A. fiavus in particular) under various simulated seasonal conditions of storage, particularly in the Natal province.
Africa. Having obtained ethical and parental consent for the study, patients were classified as cases of kwashiorkor, marasmus or underweight (Wellcome classification). Well nourished age-matched children who had arrived at the hospital and were being investigated for other conditions were taken as controls. For every one or two malnourished patients a control child of the same age was selected. Altogether, 109 consecutive patients were admitted to the study. Blood for aflatoxin analysis was taken in the Outpatients Department on the day of admission. Collection of a urine sample, however, was not always possible. All samples were kept frozen prior to analysis. Aflatoxin analysis was carried out by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) using fluorescence detection." Analysis of serum was performed after hexane partitioning, chloroform extraction and silica-column clean-up. Detection of major aflatoxins Bp B 2, G" G 2 and M 1 was facilitated using the above methodology. The lower limits of detection for aflatoxins (pg/ml) were as follows: serum: B" 25; B2 , 1; Gp 50; G 2 , 1; M" 5; urine: B" 25; B2 , 10; G" 25; G 2, 10; Mp 5.
Methods Seed storage Mycofiora pilot study
Newly harvested maize seeds were stored in sterilized, sealed containers under various conditions of temperature and relative humidity (RH) to simulate the seasons of autumn and summer in Natal, Transvaal winter (TW) and Natal winter (NW) (Table I). Seeds were tested for fungal contamination and moisture content (MC) at the outset and after 3, 6, 9 and 12 weeks of storage.
Patients Children between the ages of 6 months and 2 years with malnutrition were selected from the Paediatric Outpatient Department of King Edward VIII Hospital, Durban, South
Statistical analysis Chi-square was used with Yates' correction. If an expected cell value was < 5 then Fisher's Exact test was used.
Results Seed-storage mycofiora (Figs. 1a, 1b, 2a, 2b)
Fungal contamination prior to storage (0 time) showed that a mixed mycoflora comprising Fusarium spp. ( 100%) and Penicillium spp. (94%) dominated the seeds' internal environment. Proliferation of Aspergillus spp. generally was evident throughout the storage period. However, under summer and autumn conditions (Figs. 1a, 1b) proliferation of A. fiavus
Aflatoxins and kwashiorkor in South Africa
"0 Q)
ti Q) ~
120
I
D
Fusarium spp.
Other Aspergilli
~ A. ochraceus
Penicillium spp.
-
Moisture content
A. flavus gp. spp
243
A.
"' "' af!. "0 Q) Q)
"0 Q)
ti Q)
#
~
(/)
CD CD
"' "' af!.
c.
"0 Q) Q)
~ ....
0
0
3
6
9
12
Time in weeks
FIG. 1. Storage of maize seeds. A. Summer, B. Autumn.
and A. parasiticus (presently considered together as both are group species of A. flavus), which was negligible after 3 and 6 weeks of storage, peaked at 100% at 9 weeks, declining to an insignificant degree thereafter. These storage conditions were: 25°C and 90% RH, and alternating between 25°C by day and 18°C at night, also at 90% RH, respectively. However, storage under generally cooler conditions (NW [Fig. 2a]) and (TW [Fig. 2b]) was correlated with a low but persistent (especially under the latter conditions) incidence of A.flavus spp., although moisture content was relatively high. Under NW conditions, seed moisture was not consistently at a high level, permitting A.flavus spp. to proliferate, 12 which might explain its more erratic isolation compared with that of seeds from the TW regimes. Other fungal species of A. ochraceus, A. glaucus and A. tirreus (classified as 'other Aspergilli'), Penicillium spp. and Fusarium spp. were also predominant under all storage conditions (Figs. 1a, band 2a, b).
Results Serum analysis Serum analysis for aflatoxin was carried out on controls (35), marasmic ( 13), underweight (16) and kwashiorkor (45) children ranging in age from 6 months to 2 years. Aflatoxins were detected in the serum of 56% of both kwashiorkor and underweight cases, 31% of those with marasmus, and 49% of control infants. There were no significant differences among the various groups in the percentage of aflatoxin-positive results (Table II).
Individual serum aflatoxins Prevalence. Serum from the kwashiorkor group showed a greater prevalence of the individual aflatoxins than that from the other groups, aflatoxin B1 (18%), aflatoxin B2 (27%) and aflatoxin G 2 (20%) being detected more frequently. There was a consistent presence of aflatoxin M 1 in all groups except the marasmic. The mean percentage
244
G. Ramjee et al.
Ill A. flavus gp. spp -g
120
t>
~ Other Aspergilli
[ ] Fusarium spp. ~ A. ochraceus
I§!S Penicillium spp.
-
A. 30
Moisture content
Q)
1; rn
20
80
3:::
"0 Q) Q)
rn Q) ~ rn
"0
"'
80
3:::
Q) Q)
0
rn
