Determinants of PCR performance (Xpert MTB/RIF), including bacterial load and inhibition, for TB diagnosis using specimens from different body compartments

Abstract

The determinants of Xpert MTB/RIF sensitivity, a widely used PCR test for the diagnosis of tuberculosis (TB) are poorly understood. We compared culture time-to-positivity (TTP; a surrogate of bacterial load), MTB/RIF TB-specific and internal positive control (IPC)-specific C(T) values, and clinical characteristics in patients with suspected TB who provided expectorated (n = 438) or induced sputum (n = 128), tracheal aspirates (n = 71), bronchoalveolar lavage fluid (n = 152), pleural fluid (n = 76), cerebral spinal fluid (CSF; n = 152), pericardial fluid (n = 131), or urine (n = 173) specimens. Median bacterial load (TTP in days) was the strongest associate of MTB/RIF positivity in each fluid. TTP correlated with C(T) values in pulmonary specimens but not extrapulmonary specimens (Spearman's coefficient 0.5043 versus 0.1437; p = 0.030). Inhibition affected a greater proportion of pulmonary specimens than extrapulmonary specimens (IPC C(T) > 34: 6% (47/731) versus 1% (4/381; p < 0.0001). Pulmonary specimens had greater load than extrapulmonary specimens [TTPs (interquartile range) of 11 (7-16) versus 22 (18-33.5) days; p < 0.0001]. HIV-infection was associated with a decreased likelihood of MTB/RIF-positivity in pulmonary specimens but an increased likelihood in extrapulmonary specimens. Mycobacterial load, which displays significant variation across different body compartments, is the main determinant of MTB/RIF-positivity rather than PCR inhibition. MTB/RIF C(T) is a poor surrogate of load in extrapulmonary specimens.

Figure 1. Comparison of culture days-to-positivity (A and B) and Xpert MTB/RIF C_T values (C and D) in different fluids, and in pulmonary specimens versus extrapulmonary specimens.

Asterisks indicate specimen types with significantly lower bacterial load than expectorated sputum or pulmonary specimens. Abbreviations: BALF, bronchoalveolar lavage fluid; CSF, cerebral spinal fluid. No urine specimens were culture-positive.

Figure 2. Scatter plots of days-to-positivity in different types of liquid culture-positive specimens obtained from separate patient cohorts.

Each circle represents an individual specimen. Solid circles indicate specimens which were Xpert MTB/RIF-positive, whereas empty circles indicate Xpert MTB/RIF-negative specimens. Comparisons below each graph are between median (IQR) TTPs for Xpert-MTB/RIF-positive vs. –negative specimens for that fluid. *Only one MTB/RIF-negative, culture-positive tracheal aspirate specimen was present; Fluids from the lung include expectorated sputum, induced sputum, tracheal aspirates, and bronchoalveolar lavage fluid; Fluids from elsewhere in the body include pleural fluid, cerebral spinal fluid, and pericardial fluid. No patients had culture-positive urine.

Figure 3. Scatter plots of Xpert MTB/RIF internal positive control (IPC) cycle threshold values in different types of culture-positive specimens obtained from separate patient cohorts.

Each circle represents an individual specimen. Solid circles indicate patients who were Xpert MTB/RIF-positive, whereas empty circles indicate Xpert MTB/RIF-negative specimens. Comparisons are between median (IQR) IPC C_Ts for Xpert-MTB/RIF-positive vs. –negative specimens. *Only one MTB/RIF-negative, culture-positive tracheal aspirate specimen and one MTB/RIF-negative, culture-positive BALF specimen were present; Pulmonary specimens include expectorated sputum, induced sputum and bronchoalveolar lavage fluid; Extrapulmonary specimens include tracheal aspirates, pleural fluid, cerebral spinal fluid, and pericardial fluid. No patients had culture-positive urine.

Figure 4. Correlation between liquid culture time-to-positivity and Xpert MTB/RIF cycle threshold values in (A) pulmonary specimens and (B) extrapulmonary specimens.