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Influence of host factors, including human immunodeficiency virus (HIV) co-infection, on the distribution and diagnostic potential of previously evaluated biomarkers of pulmonary tuberculosis (PTB), such as anti-antigen 60 (A60) immunoglobulin (Ig) G, anti-A60 IgA, and C-reactive protein (CRP), remain unclear. Anti-A60 IgG, anti-A60 IgA, and CRP in PTB and non-PTB patient sera (n = 404, including smear-positive/negative, culture-positive (SPCP/SNCP) and HIV+ve/-ve) were measured by enzyme-linked immunoassay and statistically analysed. In multinomial logistic regression, expectoration, chest pain, wasting, and culture count positively associated with CRP (p < 0.001), while smear count positively associated with anti-A60 IgG (p = 0.090). Expectoration and enlarged lymph nodes negatively associated with anti-A60 IgA (p = 0.018). Biomarker distribution and diagnostic potential varied significantly by symptoms and bacilli burden, and across different PTB subpopulations. CRP was correlated poorly with anti-A60 antibodies, while anti-A60 IgA and IgG were correlated in non-tuberculosis (TB) and SPCP patients (p < 0.001). When combined, anti-A60 IgG and CRP best discriminated SPCP/HIV-ve from non-TB (AUC: 0.838, 95% CI: 0.783(-)0.894), while anti-A60 IgA and CRP performed best in discriminating HIV+ve PTB from non-TB (AUC: 0.687, 95% CI: 0.598(-)0.777). Combined CRP and anti-A60 antibodies had significantly reduced accuracy in SNCP and SNCP/HIV+ve compared to SPCP/HIV-ve subpopulations. The complex relationships between host factors and biomarkers suggest their limited utility, especially in SNCP/HIV+ve subpopulations, highlighting the importance of examining host response and immune biomarkers across relevant patient subpopulations.