Diagnosis of Tuberculosis through qPCR Amplification of Mycobacterium tuberculosis DNA in Patient Urine Samples

Abstract

Mycobacterium tuberculosis (Mtb) is one of the most widespread bacterial pathogens, having infected about 25% of the world’s population. Mtb is spread through aerosols and can remain dormant in infected individuals for years. The disease is especially prevalent in developing countries in Africa, which have a tuberculosis incidence rate ten times that of Europe. Current sampling methods make diagnosis difficult in these regions. The common skin test has low accuracy and does not identify for the strain or progression of the disease. Other methods such as examination of sputum smears, sputum culture, and GeneXpert require the collection of sputum which must be refrigerated and quickly transported to the analysis site. Infants and young children do not understand how to provide a sputum sample. Urine is a potentially more stable and practical diagnostic fluid. Protein-based urine MTb tests have been effective, but require trained personnel, and refrigerated transport. The antibody-based diagnosis cannot readily identify drug resistance, unlike DNA-based diagnostic tests. The purpose of our study was to determine if urine spiked with Mtb DNA can be detected by qPCR. If so, can Mtb DNA from TB+ patients be identified in their urine via qPCR testing? Urine samples were obtained via an IRB-approved Mason study. Six Guinea-Bisseau children ages 6-12 diagnosed with TB by other methods were amplified through qPCR using rpoB forward and reverse primers. The amplification curves produced were compared to a standard curve of genomic DNA from Mtb strain H37Ra to determine the concentration of copies in patient urine. Three replicates of six samples were tested, with half of the trials showing detectable levels of amplification. Three of these samples were amplified on at least one replicate but not all three replicates. in contrast, all MTb-spiked control urine replicates were amplified. This is consistent with our previous study using PCR. It is possible that Mtb DNA in patient samples may be physically or biochemically inaccessible for qPCR analysis. Future studies may identify procedures for more consistent diagnostic results in patient urine samples.