Multiplex PCR and quality control of Epinotia aporema granulovirus production.

A specific multiplex PCR was developed for the rapid and highly sensitive quality control of the viral DNA during Epinotia aporema granulovirus (EpapGV) production. At the beginning of this work only 2.3% of the EpapGV genomic sequence was known. In order to increase the availability of specific information, the terminal sequences of the inserts of several selected clones of EpapGV genomic libraries were determined. These data comprised 8.4% of the total DNA sequence and corresponded to regions distributed throughout the genome. Based on the small fraction of known sequence available a set of 32 primers was designed, using information theory to set the basis for this study. Each pair of designed primers was initially tested in individual PCRs to assess the correct size of the expected product and the sensitivity of the amplification. The specificity was verified in multiplex PCRs, using alternatively 1-3 sets of selected 5-6 primer pairs and EpapGV DNA preparations from different sources and degrees of purity. The results indicate that the multiplex PCR could be used for quality control in the bioinsecticide production, as well as in other applications such as the detection of latent infections in E. aporema colonies, and studies related to virus distribution, vertical transmission, host range, or persistence in the field.

Processing of cassava roots to remove cyanogens: short communication

A simple equation is developed between the total cyanide contents of cassava root parenchyma and the processed product with the percentage retention of cyanide on processing. This equation is applied to different methods of processing used worldwide. Thus to produce cassava flour to 10 mg HCN equivalents/kg flour (PPM); the WHO safe level; by sun drying or heap fermentation requires starting with sweet cassava containing 12-32 ppm total cyanide. In an average year only 14 per cent of flour samples in our study areas in Nampula Province of Mozambique had total cyanide contents of 10 ppm. Distribution curves of flour total cyanide show that the percentage of samples exceeding 100 ppm total cyanide increased from 6 per cent in an average year to 43-65 per cent in a low rainfall year; when cases of konzo also occurred. Processing methods used to produce farinha in Brazil and gari in West Africa reduce the total cyanide content to less than one eighth of that using heap fermentation and less than one sixteenth of that using sun drying. Heap fermentation and sun drying; commonly used in eastern and southern Africa; do not adequately remove cyanide in a normal year and are hopeless inadequate when used on cassava grown during drought. New and greatly improved processing methods are urgently needed. The high levels of cyanide intake in central; eastern and southern Africa from high cyanide flour are most likely cause of konzo in young people and the very long term consumption of gari of lower cyanide content in West Africa is the most likely cause of TAN in order people

Combination of cassava flour cyanide and urinary thiocyanate measurements of school children in Mozambique.

The maximum daily cassava flour intake of children may be calculated from determination of the total cyanide content of cassava flour and urinary thiocyanate levels of school children in samples collected at the same time and place. Four sites, two with and two without recent konzo cases, were chosen for study. In two sites with recent konzo cases, 84% and 93% of school children consumed cassava the previous day, and the calculated maximum daily consumption of cassava was over 700 g. In two sites without recent konzo cases, about 50% of school children consumed cassava the previous day and the calculated daily consumption of cassava flour was less than 150 g. By measurements of cyanide in flour and urinary thiocyanate we are therefore able to distinguish between communities whose diet is almost totally reliant on cassava, and who are therefore susceptible to konzo, and those who have a broader diet and are free from konzo. In another calculation it is shown that 4-23% of the essential S-containing amino acids in the cassava flour consumed by children is used up to detoxify and flour cyanide to thiocyanate. This depletion of methionine and cystine may leads to protein deficiency and may contribute to onset of konzo.

Persistent konzo and cyanogen toxicity from cassava in northern Mozambique.

We aimed to detect new cases of konzo and monitor cyanogen exposure from cassava flour in communities previously affected by konzo epidemics in Nampula Province, northern Mozambique. Other objectives were to detect subclinical upper motor neuron damage in schoolchildren and test a new kit to measure urinary thiocyanate concentration. In 1999 and 2000, we carried out active and passive case detection for konzo in Memba and Mogincual Districts. In July and October, 1999, we collected cassava flour from 30 houses in three communities and measured cyanogen concentrations with a picrate kit. In October 1999, we examined all schoolchildren in three communities for ankle clonus and measured urinary thiocyanate concentration in thirty schoolchildren in each of five communities with a picrate kit. We found 27 new cases of konzo in Mogincual District. Mean total cyanogen concentrations in cassava flour varied between both seasons and years, but were always high, ranging from 26 to 186 ppm. Very high mean levels at three sites in November 1998 and July 1999 were probably due to low rainfall in the 1997-1998 season. The proportion of schoolchildren with ankle clonus varied from 8 to 17%. The new picrate kit for urinary thiocyanate worked well; mean concentrations in schoolchildren ranged from 225 to 384 micromol x l(-1). Konzo and sub-clinical upper motor neuron damage persist in poor rural communities in northern Mozambique, associated with high cyanogen concentrations in cassava flour and high urinary thiocyanate concentrations in schoolchildren.