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

Abstract

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...

A recent update of the diagnostic methods for tuberculosis and their applicability in Indonesia: a narrative review

“…Lastly, the specimen is incubated at 37°C for 6 weeks. 17,23 Culturing of M. tuberculosis is effective at 10-10 2 CFU/ml and has a higher sensitivity than AFB staining. However, the growth of M. tuberculosis in culture is very slow.…”

“…The Ct values are classified into high (<16), medium (16)(17)(18)(19)(20)(21)(22), low (22)(23)(24)(25)(26)(27)(28), and very low (>28) categories.⁶…”

“…22,28 Then, 2 ml of the mixture is inserted into the cartridge and the PCR is run automatically to detect M. tuberculosis and rifampicin resistance. 17 In pulmonary TB, the assay can be performed using either raw sputum or pellet sputum. 5,18 The GeneXpert assay may also produce falsepositive or false-negative results.…”

“…A major challenge for TB diagnosis using conventional methods is their poor sensitivity in detecting M. tuberculosis (20-80%). [16][17][18] The diagnosis of EPTB is even more challenging due to the low number of bacteria in the specimens taken from infected sites.¹⁹ Culturing is the standard reference to diagnose TB, but this requires 2-8 weeks. 20,21 Further details on TB diagnosis and the challenges are discussed below.…”

Evaluasi MetodeGeneXpert MTB/RIF dengan SampelRaw Sputum untuk Mendeteksi Tuberkulosis Paru

“…3 Tantangan utama dalam mendiagnosis TB secara konvensional adalah rendahnya sensitivitas deteksi pada pemeriksaan mikroskopis, yaitu sebesar 20-80%, dan lamanya waktu yang diperlukan untuk mendapatkan hasil kultur. 3,4 Meskipun merupakan baku emas untuk diagnosis TB, diperlukan 2-8 minggu untuk melakukan kultur dengan infrastruktur yang tepat dan tenaga ahli yang profesional. 5,6 GeneXpert MTB/RIF assay (Cepheid, Sunnyvale USA) merupakan metode untuk mendeteksi 18 Penelitian ini mempunyai keterbatasan seperti pemrosesan sampel yang tidak dilihat secara langsung oleh peneliti dan tidak adanya data riwayat penyakit dahulu serta riwayat pengobatan pasien.…”