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

Theron, Grant; Peter, Jonny; Calligaro, Greg; Meldau, Richard; Hanrahan, Colleen F.; Khalfey, Hoosain; Matinyenya, Brian; Muchinga, Tapuwa E.; Smith, Liezel; Pandie, Shaheen; Lenders, Laura; Patel, Vinod; Mayosi, Bongani M.; Dheda, Keertan

doi:10.1038/srep05658

Cited by 116 publications

(103 citation statements)

References 44 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Clinical Substudy (I) Clinical Study Participantsmentioning

confidence: 99%

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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

et al. 2015

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The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection of Mycobacterium tuberculosis in sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions of M. tuberculosis into sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained from M. tuberculosis culture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in which M. tuberculosis was detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%; P ‫؍‬ 0.03) and for smear-negative specimens (67% versus 58%; P ‫؍‬ 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increased M. tuberculosis detection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.A major challenge to the reduction of the global burden of tuberculosis (TB) (1) has been the poor sensitivity and timeliness of conventional diagnostic tests, such as smear microscopy and mycobacterial culture. The Xpert MTB/RIF (Xpert) assay is a PCR-based diagnostic that detects Mycobacterium tuberculosis and rifampin (RIF) resistance within 2 h (2). Xpert was endorsed by the WHO in December 2010 for use in high-burden countries and is the first rapid near-point-of-care diagnostic to be widely implemented in TB-endemic settings (3). In clinical studies among adults, the sensitivity of the Xpert assay was 95 to 100% in smearpositive, culture-positive TB cases and 43 to 100% in smear-negative, culture-positive TB cases; specificity was 99 to 100% (2, 4-6).The Xpert assay procedure for the detection of M. tuberculosis from sputum has been well described (2, 5). In brief, the sputum sample is mixed with a "sample reagent" (SR) that is used to liquefy the sample, reduce biohazard, and inactivate PCR inhibitors. Two milliliters of the mixture is placed into the Xpert cartridge, and the cartridge is inserted into the GeneXpert instrument, where fully automated PCR is completed to detect both M. tuberculosis and RIF resistance (2, 5). An estimate of the M. tuberculosis concentration in the sample is provided by the threshold cy...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Clinical Substudy (I) Clinical Study Participantsmentioning

confidence: 99%

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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

et al. 2015

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“…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).…”

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confidence: 99%

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

et al. 2016

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A systematic review investigating the role of Xpert MTB/RIF in the diagnosis of tuberculous pleural effusion (TPE) was conducted. The pooled sensitivities and specificities of Xpert MTB/RIF were 51.4% and 98.6%, respectively, with culture used as a reference standard and 22.7% and 99.8%, respectively, with a composite reference standard (CRS) used as the benchmark. Xpert MTB/RIF has low sensitivity but excellent specificity in the diagnosis of TPE.T uberculous pleural effusion (TPE) is the second most common site of extrapulmonary tuberculosis (EPTB) (1). Currently, the best tool for diagnosing TPE is a thoracoscopic pleural biopsy, an invasive procedure, with sensitivity ranging from 93 to 100% (2-5). The most widely used diagnostic test for TPE is the pleural fluid adenosine deaminase (ADA) level, which has variable sensitivity ranging from 47 to 100% (6). The sensitivities of pleural fluid microscopy and culture in TPE are about 10% and 20%, respectively (5, 7, 8). The absence of a simple reference standard makes the treatment of TPE empirical (nonmicrobiological) in most circumstances.Xpert MTB/RIF (GeneXpert) has not only high sensitivity and specificity in smear-positive pulmonary tuberculosis (TB) (98% and 98%, respectively) but also reasonable diagnostic performance in smear-negative pulmonary tuberculosis (67% and 99%, respectively) (9). However, a recent meta-analysis reported the pooled sensitivity and specificity of GeneXpert in TPE as 46.4% and 99.1%, respectively, compared with those of pleural fluid mycobacterial culture (10). Since then, several new studies evaluating GeneXpert in TPE have been published. Herein, we perform an updated meta-analysis on the role of Xpert MTB/RIF in the diagnosis of TPE.The systematic review was conducted using the standard methodology for systematic reviews of diagnostic test accuracy (see the detailed methodology in the supplemental material) (11,12). Institutional ethics committee approval was not required as this was a systematic review of published data.Our search yielded 155 citations, of which 24 studies (2,486 patients) were included in the current analysis (see Fig. S1 in the supplemental material) (7,8,. There were equal numbers of studies from the developed and the developing countries (see Tables S1 and S2 in the supplemental material) (8, 14, 16-22, 24, 32, 33). 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). Seven studies performed Xpert MTB/RIF on frozen pleural fluid (7,15,19,21,23,28,30), while the remaining used fresh samples (see Table S4). Nine studies concentrated the pleural fluid sample by centrifugation before performance of GeneXpert (7, 8, 16-18, 20, 22, 24, 29). The ov...

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Erer¹,

Erol²,

Anar³

et al. 2017

Turk J Med Sci

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

Cited by 116 publications

References 44 publications

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

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

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

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

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