Quantification of IgG in Lymphocyte Supernatant Assay as a biomarker of pediatric TB
T. Sattar1, K. Ahmed 1, A. Tariq 1, Z. Mujahid1, A. Ansari1, F. Qamar 1, N.T. Iqbal1,2
1 Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan, 2 Department of Biological and Biomedical Sciences, Aga Khan University, Karachi.
Background: Pakistan ranks 6th in the top 30 highest TB burden countries. Amongst all cases reported in the year 2019, 13% of cases were reported in children. A majority of pediatric population remain undiagnosed due to non-standardized scoring criteria, and non-availability of sensitive diagnostic tests. Previously, Antibody in Lymphocyte Supernatant (ALS) assay was performed in pediatric population with sensitivity and specificity of 78% and 86% respectively. To enhance the sensitivity of ALS assay enrichment of CD19+B cells from PBMCs isolation was carried out for the quantification of B cells in ELISA.
Methods: In this study, GeneXpert +ve pediatric TB (cases=48) aged between 1-18 years and age and sex matched (controls=49) were enrolled. B cells were harvested by using CD19 (Dynabeads) from PBMCs and cultured unstimulated. The IgG antibody secretion within the culture supernatants were measured by ELISA using BCG vaccine and Mtb Sonicate antigens.
Results: We observed a higher response of IgG in supernatant from TB cases compared to healthy controls at 48 hrs with BCG antigen (case=0.7269±0.4922; control=0.4423±0.300, p=0.0014 MWU test). Sorting of CD19+ B cells showed a similar higher response in cases (BCG=0.1480, SON=0.114) vs. controls (BCG=0.072; SON=0.075; p=0.003, p=0.02).
Conclusion: Our findings indicates that ALS test can be used as a discriminatory biomarker for the diagnosis of active TB infection in children which can help in prompt management, and prevention of complications that eventually reduce the disease burden and morbidity. The modification in the existing ALS assay has significantly improved the sensitivity but moderately increased the specificity. These findings need to be further validated in larger longitudinal cohort studies.
1. WHO. World Health Organization. Global Tuberculosis Report. 2019:1-270.
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