Diagnostic and Treatment Monitoring Potential of A Stool-Based Quantitative Polymerase Chain Reaction Assay for Pulmonary Tuberculosis

Abstract

A quantifiable, stool-based, Mycobacterium tuberculosis (Mtb) test has potential complementary value to respiratory specimens. Limit of detection (LOD) was determined by spiking control stool. Clinical test performance was evaluated in a cohort with pulmonary tuberculosis (TB) (N = 166) and asymptomatic household TB child contacts (N = 105). Stool-quantitative polymerase chain reaction (qPCR) results were compared with sputum acid-fast bacilli (AFB) microscopy, GeneXpert MTB/RIF (Xpert MTB/RIF), and cultures. In Mtb stool-spiking studies, the LOD was 96 colony-forming units/50 mg of stool (95% confidence interval [CI]: 84.8-105.6). Among specimens collected within 72 hours of antituberculosis treatment (ATT) initiation, stool qPCR detected 22 of 23 (95%) of culture-positive cases. Among clinically diagnosed cases that were Xpert MTB/RIF and culture negative, stool qPCR detected an additional 8% (3/37). Among asymptomatic, recently TB-exposed participants, stool PCR detected Mtb in two of 105 (1.9%) patients. Two months after ATT, the Mtb quantitative burden in femtogram per microliters decreased (Wilcoxon signed-rank P < 0.001) and persistent positive stool PCR was associated with treatment failure or drug resistance (relative risk 2.8, CI: 1.2-6.5; P = 0.012). Stool-based qPCR is a promising complementary technique to sputum-based diagnosis. It detects and quantifies low levels of stool Mtb DNA, thereby supporting adjunct diagnosis and treatment monitoring in pulmonary TB.

Figure 1.

Cohort enrollment. TB = tuberculosis; PCR = polymerase chain reaction.

Figure 2.

Quantity of Mycobacterium tuberculosis (Mtb) DNA detected by polymerase chain reaction (PCR) correlates with known colony-forming units (CFUs) of Mtb. Stool from a healthy control without tuberculosis was spiked with 10-fold dilutions of H37Rv Mtb (N = 6 per concentration, N = 60 total). (A) Stool DNA was isolated and Mtb qPCR quantified (Pearson’s r = 0.998, P value < 0.0001). (B) The percent of assays that detected Mtb was plotted for each concentration. Using logistic regression, there was a 95% probability of detecting Mtb in samples containing at least 95.24 CFU/50 mg of stool (95% confidence interval [CI]: 84.87–105.6) (95% CI shown in pink). This figure appears in color at www.ajtmh.org.

Figure 3.

Mycobacterium tuberculosis (Mtb) burden decreases during antituberculosis treatment (ATT). Individuals with tuberculosis provided stool samples at time of enrollment and again at the 2-month follow-up visit. (A) In most participants with treatment success, quantified Mtb polymerase chain reaction (PCR) on paired samples demonstrated a decrease in time as ATT increases (inverse correlation). (B) Individuals with treatment failure or drug resistance had an increased risk of persistent stool Mtb PCR at 2 months (relative risk 2.8 confidence interval: 1.2–6.5 P = 0.012). Rx = treatment.

Figure 4.

Quantitative Mycobacterium tuberculosis (Mtb) levels are similar despite HIV status. Individuals with tuberculosis completed stool Mtb quantitative polymerase chain reaction. Results were compared between individuals with and without HIV infection, and quantitative Mtb femtogram per microliters were similar among HIV-infected and HIV-infected individuals and did not vary by CD4 count (Kruskal–Wallis test, P value = 0.493). Error bars depict the median and interquartile range.