Five years of screening for galactosaemia in South Africa: Pitfalls of using Benedict’s test and thin layer chromatography to screen for galactosaemia in a developing country
Introduction
Galactosaemia is one of the inherited disorders of carbohydrate metabolism, which can become life threatening if not promptly diagnosed and treated. The condition arises from defects in the metabolism of galactose in the Leloir pathway which is comprised of Galactokinase(GALK), galactose-1-phosphate uridyl transferase (GALT) and galactose epimerase (GALE). GALT deficiency, the commonest type leading to classic galactosaemia [1], is associated with profound morbidity as a result of accumulation of galactose-1-phosphate, a proven toxic metabolite in various cells [2]. The associated morbidity from clinical manifestations and complications of galactosaemia can be prevented by early diagnosis and treatment. One modality of early detection is through newborn screening.
There is no formal national newborn screening policy in South Africa for inborn errors of metabolism including galactosaemia. In place of formal screening, there is selective testing done in high-risk patients in various settings and in most cases this preliminary testing is performed in patients who are already symptomatic. This rudimentary screening for galactosaemia by analysis of urine reducing substances with Benedict’s test in conjunction with thin layer chromatography (TLC) was introduced decades ago as quick, cost effective and non-laborious methods [3], [4]. Benedict’s test uses cupric sulphate which is reduced to cuprous oxide in the presence of urine reducing substances which can then be identified on thin layer chromatography. TLC is based on separation through capillary action facilitated by the mobile phase and the relative polarity of the reducing sugars [3], [4], [5].
In most developing nations, these methods remain the commonly utilised methods for the screening of hereditary galactosaemia and for the presence of galactosuria in infants [6]. However, there is limited published data on the effectiveness of testing for urine reducing substances in screening for galactosaemia and the few existing publications have been on small scale studies and case reports with conflicting findings [3], [7], [8], [9], [10]. The largest published study analysed 383 patients [6]. The aim of the study was to investigate the effectiveness of screening for hereditary galactosaemia with Benedict’s test and thin layer chromatography in a tertiary laboratory in a population with a high carrier frequency (1/14 400–1/22 500) [11] compared to the 1/30 000–1/50 000 in European populations. In our view, this is the first study of its kind to investigate on a larger scale (close to 900 subjects) the effectiveness of Benedict’s test and thin layer chromatography for galactosaemia screening.
Section snippets
Patients
This was a retrospective analysis of test requests for urine reducing substances and thin layer chromatography from patients during the period 2013 – 2017. The patient results were derived from four inland provinces, with a total population of approximately 15 million, served by the laboratory. In addition, results of the confirmatory tests i.e. Galactose-1-phosphate uridyltransferase (GALT) activity and DNA studies were also analysed (Fig. 1).
Benedict’s test
Reagent tablets (AimTab™ reducing substances
Results
In the 5-year period, 878 paediatric patients (5 days −12 years) were screened using the Benedict’s test (Tables 1 and 2; Fig. 1). A moderately higher proportion of the screened population were males at 58%. We also had 9 older subjects with ages from 13 to 19 years. The combined ages of the subjects at the time of the screening ranged from 5 days to 19 years with a median age of 52 days. 48% tested positive/trace on the Benedict’s test of which 52% of these had galactose present on thin layer
Discussion
GALT-deficient classical galactosaemia is the commonest inborn error of carbohydrate metabolism resulting in significant morbidity and mortality if not promptly detected and treated. This has been a comprehensive study in the investigation of the use of Benedict’s test and thin layer chromatography for the screening of galactosaemia in a setting of high incidence [11]. The study covered a wide population from four inland provinces. The results of this study concur with the findings of previous
Conclusion
Galactosaemia is a potentially manageable carbohydrate metabolic disorder if treated early in infancy to prevent immediate and severe complications. In developing nations, where screening is done using Benedict’s test and TLC, based on our research findings, it is recommended that a negative Benedict’s test in a clinically suspicious patient should be repeated after a trial of lactose loading. It should also be born in mind that false positive cases may arise from liver pathology (a
CRediT authorship contribution statement
Tumelo M. Satekge: Conceptualization, Investigation, Writing - original draft. Olivia Kiabilua: Conceptualization, Methodology. Amanda Krause: Resources, Data curation, Writing - review & editing. Tahir S. Pillay: Writing - review & editing, Visualization, Project administration.
Acknowledgements
We appreciate the assistance of Dr L. Bhengu and Mr I. Sinclair for data extraction. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. This work was submitted in fulfilment of the MMed (Chem Path) degree dissertation requirements for T.M. Satekge at the University of Pretoria.
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