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 CT 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 CT 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 CT > 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 CT is a poor surrogate of load in extrapulmonary specimens.

Improving the Sensitivity of the Xpert MTB/RIF Assay on Sputum Pellets by Decreasing the Amount of Added Sample Reagent: a Laboratory and Clinical Evaluation

“…These findings support further Overdilution of clinical samples prior to Xpert testing could lead to a decreased concentration of M. tuberculosis bacilli in the Xpert cartridge and therefore lead to lower detection. In fact, lower sensitivity of Xpert for detection of M. tuberculosis has been reported for sputum samples that have been induced with saline than for those that were expectorated (13), possibly because of sample dilution. In the Cepheid package insert (8), clinical data are reported comparing Xpert sensitivity between 535 expectorated specimens and 234 induced specimens.…”

“…First, all clinical samples were obtained from HIV-positive patients who, especially those with advanced immunosuppression (15), are more likely to be sputum AFB smear negative due to a higher frequency of noncavitary pulmonary disease (13) and lower bacterial burden (16). Therefore, our results may not be relevant to HIV-negative patients, who have a higher burden of M. tuberculosis and whose sputum samples are more likely to be smear positive and to have a shorter time to growth on MGIT.…”

“…The above-mentioned results suggest that the 2:1 SR treatment ratio would be most beneficial in sputum samples with low M. tuberculosis bacterial loads. Although we did not perform quantitative CFU plating of the clinical samples, we did record the TTP for each sputum sample, which has been used as an indication of the bacillary burden in clinical samples (12)(13)(14). Of 134 samples tested in our study, three MGIT liquid cultures were contaminated with nonmycobacterial organisms and four were positive only for nontuberculous mycobacteria (these samples were confirmed to have M. tuberculosis by solid culture).…”

Diagnostic Performance of Xpert MTB/RIF in Tuberculous Pleural Effusion: Systematic Review and Meta-analysis

“…Only two studies included children (13,15). Seven studies included people living with HIV infection (8,17,18,(20)(21)(22)27). Twenty-one studies (2,167 patients) and 10 studies (937 patients) used a microbiological standard (mycobacterial culture) (7, 8, 13-16, 19, 20, 22-34) or a composite reference standard (CRS) (7, 14-18, 21, 22, 24, 28), respectively, to assess the diagnostic accuracy of GeneXpert (see Table S3).…”

The diagnostic accuracy of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in mediastinal tuberculous lymphadenitis

“…When these results are compared with fineneedle aspiration results for peripheral lymph nodes and EUS studies for mediastinal lymph nodes, sensitivities and specificities are similar (2,(18)(19)(20)(21). As a result, conventional microbiological methods have low diagnostic sensitivity for the diagnosis of MTLA, mostly due to a low burden of bacilli and insufficient sample amount (22). In recent years, NAATs, like PCR, are being used in the diagnosis of EPT.…”