Exploring Alternative Biomaterials for Diagnosis of Pulmonary Tuberculosis in HIV-Negative Patients by Use of the GeneXpert MTB/RIF Assay

Abstract

The utility of the GeneXpert MTB/RIF (Xpert) assay for detection of Mycobacterium tuberculosis in sputum samples has been extensively studied. However, the performance of the Xpert assay as applied to other readily accessible body fluids such as exhaled breath condensate (EBC), saliva, urine, and blood has not been established. We used the Xpert assay to test EBC, saliva, urine, and blood samples from HIV-negative, smear-and culture-positive pulmonary tuberculosis (TB) patients for the presence of M. tuberculosis. To compare the ability of the assay to perform bacterial load measurements on sputum samples with versus without sample processing, the assay was also performed on paired direct and processed sputum samples from each patient. The Xpert assay detected M. tuberculosis in none of the 26 EBC samples (sensitivity, 0.0%; 95% confidence interval [95% CI], 0.0%, 12.9%), 10 of the 26 saliva samples (sensitivity, 38.5%; 95% CI, 22.4%, 57.5%), 1 of 26 urine samples (sensitivity, 3.8%; 95% CI, 0.7%, 18.9%), and 2 of 24 blood samples (sensitivity, 8.3%; 95% CI, 2.3%, 25.8%). For bacterial load measurements in the different types of sputum samples, the cycle thresholds of the two M. tuberculosis-positive sputum types were well correlated (Spearman correlation of 0.834). This study demonstrates that the Xpert assay should not be routinely used to detect M. tuberculosis in EBC, saliva, urine, or blood samples from HIV-negative patients suspected of having pulmonary tuberculosis. As a test of bacterial load, the assay produced similar results when used to test direct versus processed sputum samples. Sputum remains the optimal sample type for diagnosing pulmonary tuberculosis in HIV-negative patients with the Xpert assay.

Diagnostic accuracy of nucleic acid amplification tests in urine for pulmonary tuberculosis: A meta-analysis

“…6,17 The two main gene targets explored in this review include the M. tuberculosis complex-specific insertion sequence, IS6110, which has been suggested to be the standard tool for TB diagnosis; 32 and the gene coding for the β subunit of the bacterial RNA polymerase, rpoB, employed in Xpert, which amplifies an M. tuberculosis-specific sequence of this gene and detects mutations to determine rifampicin resistance. 33 In the face of an undefined optimal gene target for urine TB NAAT, 9,10,14,15,17,18 our findings suggest that rpoB may be less sensitive than IS6110.…”

“…Duplicates were excluded and, of the remaining 154 abstracts, 29 were eligible for full text assessment. Of these, eight studies 9,10,[14][15][16][17][18][19] were included for quantitative synthesis as depicted in the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow chart (Figure 1). 20 Eligible studies were conducted between 1993 and 2013 and included 1212 participants from seven countries.…”

“…9,10,[14][15][16]18,19 Specificity for sputum-negative study participants 9,[16][17][18][19] and specificity for healthy controls [14][15][16][17][18] could each be calculated in five studies. Three of these studies included both sputum-negative participants and healthy controls.…”

“…6,17 The two main gene targets explored in this review include the M. tuberculosis complex-specific insertion sequence, IS6110, which has been suggested to be the standard tool for TB diagnosis; 32 and the gene coding for the β subunit of the bacterial RNA polymerase, rpoB, employed in Xpert, which amplifies an M. tuberculosis-specific sequence of this gene and detects mutations to determine rifampicin resistance. 33 In the face of an undefined optimal gene target for urine TB NAAT, 9,10,14,15,17,18 our findings suggest that rpoB may be less sensitive than IS6110.This calls for further analysis in future studies as the World Health Organization endorsed PCR platform, Xpert, gains wider coverage and is further studied. Data on the reproducibility of the in-house urine PCR assays employed in this review [14][15][16][17][18] are needed, as these platforms can be highly unreliable.…”

“…9,10 Cell-free nucleic acids released from dying human cells and Mycobacterium tuberculosis microorganisms may be filtered through the kidneys, resulting in the detection of trans-renal DNA fragments in urine. 6 Although the exact pathophysiological mechanisms underlying the detection of mycobacterial DNA in the urine of patients with active PTB are not well understood, it has high potential relevance for clinical practice.…”