Prevalence of sickle cell traits in samples from Western Uganda and validation of one spot restriction fragment length polymorphism assay at the Central Public Health Laboratories

Abstract

The sickle cell trait gene in population poses a future threat of sickle cell disease in children whenever there is pairing of carrier/trait parents. Most of the patients seeking sickle cell disease and trait testing are attended and diagnosed from health care facility laboratories most of which have limited or no capacity to distinguish between the sickler and trait. Dry blood spot samples collected from health facility laboratories in twenty-nine districts of Western Uganda were tested by hemoglobin electrophoresis (to determine the prevalence of sickle cell trait) at the Central Public Health Laboratories. A total of sixty samples that tested both positive and negative for sickle cell disease including already analyzed from the routine were systematically selected and further analysed by PCR technique using one spot and two spots for validation purposes. Data collected upon testing was analysed using a statistical package (SPSS Version 20.0). Of the 384 samples that were tested by haemoglobin electrophoresis, 322 (83.9%) tested normal haemoglobin (HbAA), followed by trait (HbAS and HbFAS) that were 47 (12.2%), this was followed by sickler (HbSS and HbFS) with 14 (3.6%) while the least genotype was variant (HbAV) with 1 (0.3%). The one spot PCR showed 100% sensitivity and specificity at 95% confidence interval as compared with the gold standard test (electrophoresis) and hence the two tests showed a very strong correlation. However, two spots PCR showed moderate correlation with electrophoresis and one spot PCR. Results of the kappa model showed that there is a strong level of agreement between PCR result of one spot and gold standard in the diagnosis of sickle cell disease. There is moderate level of agreement between PCR results of two Spots and the gold standard in the diagnosis of Sickle Cell disease. There was noted moderate level of agreement during sickle cell disease testing by PCR using one spot and two spots with kappa value of 0.79. The p-value = 0.001 level of significance when tested at 95% confidence interval. Based on the results of the study, the prevalence of sickle cell trait among samples from Western Uganda was 12.2%. Diagnostic accuracy of PCR based technique using one spot is as dependable as using two spots (from agreement by kappa analysis model).