Can a simple flotation method lower the limit of detection of Mycobacterium tuberculosis in extrapulmonary samples analyzed by the GeneXpert MTB/RIF assay?

Abstract

The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study, we determined the lower limit of detection (LOD) of the GeneXpert MTB/RIF assay with nonrespiratory specimens and investigated the utility of flotation procedures for concentrating the bacilli. Clinical specimens (9 cerebrospinal fluid [CSF], 13 gastric aspirate, 8 tissue, and 17 stool) were spiked with single-celled Mycobacterium tuberculosis, and the LOD of the GeneXpert assay was determined. Flotation studies were conducted with sucrose and NaCl, and the cycle thresholds of the MTB/RIF assay were compared between treated and untreated samples. There was no significant difference between the LODs of the GeneXpert assay with saline solution (median, 33 CFU/ml) and CSF (median, 25 CFU/ml) (P > 0.05) or gastric aspirate samples (median, 58 CFU/ml) (P > 0.05). The LOD with spiked tissue (median, 1,525 CFU/ml) and stool samples (median, 6,800 CFU/ml) was significantly elevated compared to that determined with saline solution (P ≤ 0.05 and ≤ 0.0005, respectively). Flotation studies with sucrose or NaCl did not consistently result in lowered cycle thresholds in stool or gastric aspirates, but a cycle reduction of >10 was achieved in two of the three pooled CSF samples. Unlike the results seen with tissue and stool samples, there was no significant PCR inhibition in the MTB/RIF assay with CSF and gastric aspirates. Although preconcentration of CSF samples with sucrose and NaCl may enhance detection of M. tuberculosis by PCR, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in paucibacillary nonrespiratory samples.

Fig 1

Comparison of lower limits of detection of the GeneXpert MTB/RIF assay in seeded samples. Clinical specimens were spiked with dilutions of single-celled suspensions of M. tuberculosis. The lower limit of detection of the GeneXpert MTB/RIF assay in CSF (n = 9), gastric aspirate (n = 13), tissue (n = 8), and stool (n = 12) samples was compared to that in saline solution (n = 13). Wilcoxon's test was used to compare differences in medians. The asterisks indicate statistically significant difference. *, P ≤ 0.05; ***, P ≤ 0.0005; ns, not significant.

Fig 2

Comparison of cycle threshold values of the GeneXpert MTB/RIF assay on stool preparations floated with sucrose and NaCl. Stool preparations were treated with the indicated concentrations of sucrose (A and C) or NaCl (B and D). Bacilli were allowed to float by gravity (A and B) or subjected to centrifugation (C and D). Symbols represent unique patient samples. Closed symbols represent the top volume of stool preparations, while open symbols represent the middle volume.

Fig 3

Comparison of cycle threshold values of the GeneXpert MTB/RIF assay on gastric aspirates floated with sucrose and NaCl. Pooled gastric aspirates were treated with the indicated concentrations of sucrose (A) or NaCl (B). Bacilli were floated by centrifugation. Symbols represent unique pooled samples. Closed symbols represent the top volume, while open symbols represent the middle volume.

Fig 4

Comparison of cycle threshold values of the GeneXpert MTB/RIF assay on CSF samples floated with sucrose and NaCl. Pooled CSF samples were treated with the indicated concentrations of sucrose (A) or NaCl (B). Bacilli were floated by centrifugation. Symbols represent unique pooled samples. Closed symbols represent the top volume, while open symbols represent the middle volume.