Widespread use of incorrect PCR ramp rate negatively impacts multidrug-resistant tuberculosis diagnosis (MTBDRplus)

Abstract

The scale-up of rapid drug resistance testing for TB is a global priority. MTBDRplus is a WHO-endorsed multidrug-resistant (MDR)-TB PCR assay with suboptimal sensitivities and high indeterminate rates on smear-negative specimens. We hypothesised that widespread use of incorrect thermocycler ramp rate (speed of temperature change between cycles) impacts performance. A global sample of 72 laboratories was surveyed. We tested 107 sputa from Xpert MTB/RIF-positive patients and, separately, dilution series of bacilli, both at the manufacturer-recommended ramp rate (2.2 °C/s) and the most frequently reported incorrect ramp rate (4.0 °C/s). Mycobacterium tuberculosis-complex DNA (TUB-band)-detection, indeterminate results, accuracy, and inter-reader variability (dilution series only) were compared. 32 respondents did a median (IQR) of 41 (20–150) assays monthly. 78% used an incorrect ramp rate. On smear-negative sputa, 2.2 °C/s vs. 4.0 °C/s improved TUB-band positivity (42/55 vs. 32/55; p = 0.042) and indeterminate rates (1/42 vs. 5/32; p = 0.039). The actionable results (not TUB-negative or indeterminate; 41/55 vs. 28/55) hence improved by 21% (95% CI: 9–35%). Widespread use of incorrect ramp rate contributes to suboptimal MTBDRplus performance on smear-negative specimens and hence limits clinical utility. The number of diagnoses (and thus the number of smear-negative patients in whom DST is possible) will improve substantially after ramp rate correction.

Frequent suboptimal thermocycler ramp rate usage negatively impacts MTBDRsl performance for second-line drug resistant tuberculosis diagnosis

“…Ramp rate assessment was done in both an in vitro dilution series and clinical sputa (Figure 1) . DNA extracted from dilution series and clinical specimens were split and compared head-to-head at the manufacturer-recommended ramp rate of 2.2°C/s or the most common suboptimal ramp rate of 4.0°C/s identified previously in a survey 6 . MTBDR sl was done on all amplified DNA per manufacturer’s instructions for use 3 .…”

“…The manufacturer recommends a ramp rate of ≤2.2°C/s, which is the speed of temperature change between PCR cycles. We previously showed Genotype MTBDR plus (MTBDR plus ; Hain Lifescience, Germany) performance, which is an assay for first-line resistance, is reduced when suboptimal thermocycler ramp rates are used, most so on smear-negative specimens 6 . These findings are incorporated into laboratory external quality assessment programmes and WHO TB laboratory training material 7 .…”

“…We hypothesised that the heterogenous and suboptimal sensitivities reported for MTBDR sl on smear-negative specimens were partly attributable to suboptimal ramp rate and sought to generate empirical evidence of this. We assessed whether laboratories that reported use of suboptimal ramp rates during our MTBDR plus evaluation 6 had switched to the manufacturer-recommended ramp rate, and what the observed effect had been.…”

Extract from used Xpert MTB/RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk

“…5 µl dCE was used for MTBDRplus and MTBDRsl each. MTBDRplus and MTBDRsl results were reported as described 24 : either actionable [TUB-band positive and determinate (gene-specific locus bands present)] or non-actionable [TUB-band negative or TUB-band positive but indeterminate (gene-specific locus band absent)]. Susceptibility calls were made for all actionable results.…”

Tuberculosis research in South Africa over the past 30 years: From bench to bedside

“…[80] We recently identified widespread suboptimal performance of the only commercial molecular test for multidrug resistance (MTBDRplus) across dozens of laboratories worldwide and, importantly, demonstrated how this can be corrected by changing the conditions used for DNA amplification. [81] This is now in the process of being incorporated into WHO external quality assessment processes for TB laboratories.…”