Implementation of large-scale pooled testing to increase rapid molecular diagnostic test coverage for tuberculosis: a retrospective evaluation

Abstract

In 2021, only 6.4 million of the 10.6 million people with tuberculosis (TB) were diagnosed and treated for the disease. Although the World Health Organization recommends initial diagnostic testing using a rapid sensitive molecular assay, only 38% of people diagnosed with TB benefited from these, due to barriers including the high cost of available assays. Pooled testing has been used as an approach to increase testing efficiency in many resource-constrained situations, such as the COVID-19 pandemic, but it has not yet been widely adopted for TB diagnostic testing. Here we report a retrospective analysis of routine pooled testing of 10,117 sputum specimens using the Xpert MTB/RIF and Xpert MTB/RIF Ultra assays that was performed from July 2020 to February 2022. Pooled testing saved 48% of assays and enabled rapid molecular testing for 4156 additional people as compared to individual testing, with 6.6% of specimens positive for TB. From an in silico analysis, the positive percent agreement of pooled testing in pools of 3 as compared with individual testing for the Xpert MTB/RIF Ultra assay was estimated as 99.4% (95% CI, 96.6% to 100%). These results support the scale-up of pooled testing for efficient TB diagnosis.

Figure 1

Flow of pooled testing for TB detection with the Xpert MTB/RIF and Xpert MTB/RIF Ultra assays at two reference laboratories (MTB: Mycobacterium tuberculosis complex; pools and individual tests with invalid results not shown). Overall, 10,117 specimens (blue) were tested in 3501 pools, with a 5257 Xpert or Ultra assays (orange), including 1756 assays run individually for each of the specimens in the 598 positive pools.

Figure 2

Passing-Bablok regression for pools of 3 for the Xpert MTB/RIF (A) and Xpert MTB/RIF Ultra (B) assays. For the Passing-Bablok regression, the dotted line is the line of equality, and the shaded area is the 95% confidence interval. (Only specimens in pools of three with only one positive and two negative results were included; 70 for Xpert and 119 for Ultra.)

Figure 3

Estimated positive percent agreement (PPA) of pooled test results for pool size of three as compared to individual test results for the Xpert MTB/RIF and Xpert MTB/RIF Ultra assays based on in silico analysis, using historical data sets. (A) Histogram of historical clinical Xpert results with cycle threshold interval 34.1–36.7 obtained from Passing-Bablok regression (from Fig. 2A), as: 36.7 = 1.02x + 1.34; with x = 34.1; 188 of 196 specimens (blue) would test positive on pooled testing with pools of 3, while 8 specimens (red) would test negative, giving a positive percent agreement of 95.9% (95%CI, 92.1–98.2%), as shown. (B) Histogram of historical clinical Ultra results, with cycle threshold interval 28.1–30.0 obtained from the Passing-Bablok regression (from Fig. 2B), as: 30.0 = 1.16x -2.6; with x = 28.1; 161 of 162 specimens (blue) would test positive on pooled testing with pools of 3 with 1 specimen (red) that would test negative, giving a positive percent agreement of 99.4% (95% CI, 96.6–100%), as shown.