Usefulness of truenat-derived DNA from extrapulmonary specimens in direct detection of drug resistant tuberculosis by line probe assay

INTRODUCTION

The rapid nucleic acid amplification test (NAAT) Truenat^™ (Molbio Diagnostics, Goa, India) allows for the quantitative detection of Mycobacterium tuberculosis (MTB) and resistance to rifampicin (RIF) in direct sputum and extrapulmonary (EP) specimens.^[1] The system uses the Trueprep^® AUTO v2 Universal Cartridge-based Sample Prep Device and the Truelab^® Real Time mini polymerase chain reaction (PCR) Analyzer, respectively, to automate the extraction and purification of deoxyribonucleic acid (DNA) and real-time PCR. Rapid NAAT has been suggested under the National Tuberculosis Elimination Program of India (NTEP) as the initial diagnostic test for the early diagnosis of extrapulmonary (EP) tuberculosis (EPTB).^[2]NAATs (GeneXpert or Truenat) offer rapid and direct RIF resistance identification in clinical specimens; however, INH and second-line drug resistance (DR) detection takes longer in EP specimens because the bulk of these specimens is paucibacillary in nature.

Due to the suboptimal sensitivity of MTBDRplus and MTBDRsl assays on smear-negative material, MTB culture growth must be obtained before moving on to DNA extraction for the first-line and second-line drugs line probe assays (LPA).^[3,4] This can take a month time or even longer.

In addition, after 6–8 weeks of incubation, smear-negative EP specimens may turn out to be culture-negative or may even be lost because culture is prone to contamination. To retry culture, repeat patient sampling would need to be undertaken, which is difficult in EPTB patients. In these circumstances, the patient’s tuberculosis (TB) treatment will continue based only on the NAAT results of RIF drug resistance testing (DRT). Even RIF DRT information is inaccessible if RIF resistance is indeterminate in NAAT. Delaying the start of drug sensitivity test (DST)-based appropriate TB therapy in these patients runs the risk of DR amplification, significant disease damage, and patient loss to follow-up.

The prevalence of INH mono resistance is 6–8% in EP TB cases according to data from studies conducted in India and around the world,^[5] which if not treated properly may drive further RIF resistance. RIF mono-resistance is 3–7%, and multi-DR (MDR) prevalence in EPTB varies from 3% to 13.5%.^[6-8] MDR can frequently cause treatment failure and fatalities.

In a study conducted in southern India to determine the proportion of patient samples that underwent the complete DR-TB diagnostic algorithm recommended under the national TB program, it was found that 27% of patient samples did not reach the TB reference laboratories for additional testing after gene Xpert. Of those samples that did reach the laboratories, 7% did not complete the DR-TB diagnostic algorithm. Non-completion of the algorithm was more common with extrapulmonary, sputum smear-negative, and RIF resistant-TB samples. Due to culture negativity in smear-negative samples or problems with specimen transportation to affiliated TB laboratories, LPA could not be performed on these samples.^[9]

An alternate molecular-based strategy is hence urgently needed in the TB care cascade to quickly detect DR to first- and second-line anti-TB drugs in direct samples and offer evidence-based TB treatment to patients because treatment for EPTB is likely to last longer depending on the site, severity, and DR.

In this report, we outline our attempt to perform LPA on truenat-derived DNA elute from EP specimens to determine DST for INH and, where necessary, for the second-line drug. We were prompted to attempt the present work to address the concerns of clinicians in determining at the earliest whether to prescribe shorter or longer MDR treatment regimens in patients with RIF resistance or indeterminate resistance in NAAT and also for patients who had an extensive disease or were at risk of DR. Therefore, the current work was not intended to prospectively evaluate that the utility of leftover DNA elutes from a truenat test for LPA testing rather is presented here as our clinical observation. Since LPA was performed on leftover DNA which is discarded in routine after truenat test, patient consent was not required and ethics approval was not needed. Further, LPA (DNA elute) results were not formally released. The results were conveyed to clinicians for patient management only for standard LPA tests performed on smear-positive samples or culture isolates when the results were available.

MATERIALS AND METHODS

All EP samples received at our laboratory under the NTEP are subjected to truenat MTB plus test, liquid culture (MGIT 960 automated system), and fluorescence microscopy for diagnosis of TB. Truenat,^[10,11] LPA,^[12,13] and liquid culture^[14] are performed as per the manufacturer’s instructions. Routinely, direct LPA is performed on DNA extracted from smear-positive specimens while indirect LPA is on culture growth from smear-negative specimens after the specimen has been decontaminated and concentrated using the NALC-NaOH procedure.

DNA elute left over from truenat MTB positive EP samples that were received at our NRL during June–September 2022 was subjected to LPA. The workflow for EP specimen processing is depicted in [Figure 1]. LPA (DNA elute) was initially conducted on two of the EP specimens at the clinician’s request, and the results were compared with those from standard indirect LPA on culture isolates. LPA on DNA elute of additional EP samples was tried only after both LPAs showed complete concordance.

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Figure 1:

Flowchart outlining the extrapulmonary sample workflow at our tuberculosis lab.

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Briefly, MTBDRplus version 2 and MTBDRsl version 2 LPAs were performed using 5 µL of DNA volume from 70 µL to 88 µL of leftover DNA elute after the truenat test was finished without any additional purification steps or processing of DNA elutes. All EP samples were subjected to MTBDRplus, whereas MTBDRsl was only carried out on samples that were RIF or INH resistant in MTBDRplus assay. DNA elutes were stored at 4°C after the truenat test until LPA on these elutes was performed.

RESULTS

Truenat test was positive for MTB in 59 EP samples received during June–September 2022.

The EP samples comprised peritoneal fluid, pleural fluid, urine, lymph node aspirate, pus from lymph node abscess, lymph node biopsy, spinal tissue biopsy, and various tissue biopsies. Patients’ ages ranged from 12 to 79 years old (26 were male, 33 were female).

On fluorescent microscopy, smears from only two samples were positive whereas 57 samples were negative for acid-fast bacilli.

First-line LPA-FLLPA (MTBDR plus) was attempted on leftover DNA elute from these 59 truenat MTB complex positive extrapulmonary samples in the present report.

DISCUSSION

In this report, we discuss our clinical experience with conducting LPA on leftover DNA that was extracted from truenat MTB-positive clinical EP specimens.

In a study by Venter et al, MTBDRplus and MTBDRsl tests were run using cartridge extract (CE) from used Xpert TB-positive cartridges. On CE from clinical sputum specimens and culture isolates, they found high frequencies of false-RIF resistance and indeterminate resistance results with MTBDRplus assay. However, MTBDRsl demonstrated low indeterminate rates and good accuracy for both drug-sensitive-TB and DR-TB specimens when the Xpert semi-quantitation category was at least “medium” or CT 24 (equivalent to 10³ CFU/mL).^[15]

In contrast to the latter study, we observed good DST accuracy with MTBDRplus assay on truenat positive DNA elutes when the MTB count in truenat was at least 7.8 × 10² CFU/mL.

The SLLPA yield was however relatively low (11.1%) in this report although SLLPA was performed only on 9 first-line DR samples; hence, it is difficult to draw conclusions about its performance on DNA elutes.

It is noteworthy that out of 59 samples, only 21 yielded MTB-positive cultures, while the remaining 38 samples (64.4%) were lost because either the cultures were contaminated (7 samples) or growth negative (31 samples). In contrast, LPA (DNA elute) findings were obtained for 23 of these 38 culture-negative samples (60.5%), for which otherwise DST for RIF in truenat indeterminate samples and INH DST could not have been determined.

Further, time to positivity for MTB growth in liquid culture was 18–35 days, and the turnaround time (TAT) for indirect LPA from MTB culture isolates was 21–38 days long. In contrast, TAT for LPA results from DNA elutes after truenat testing was only 1–5 days.

CONCLUSION

Our results show that truenat-derived DNA elute can be a significant sample by-product that can (a) aid in detecting LPA DST in 60% of culture-negative EP specimens with MTB count of 7.8 × 10² CFU/mL or more and (b) significantly shorten the time for determination of MTB DST for the most critical drugs for TB treatment – RIF and isoniazid (INH)-in direct EP samples (within 1–5 days). Appropriate TB treatment may be otherwise delayed in these patients by almost a month or more by routine indirect LPA on EP samples. (c) In addition, it can offer potentially reliable RIF DRT results in 65% of samples that were truenat RIF indeterminate.

As far as we are aware, this is the first report of LPA performed on truenat-extracted DNA from direct EP specimens, which otherwise would have been discarded following the truenat test.

In programmatic circumstances, prompt delivery of specimens following NAAT testing to the affiliated TB laboratories for LPA, culture, and DST is crucial for the accuracy and timeliness of the diagnosis. The field facts, as seen in the study,^[4] and our everyday experience, however, indicate that transit delays of specimens or their failure to arrive at the laboratory are not unusual. (d) Since DNA is more durable than bacilli during transportation and storage, transporting DNA elute with at least MTB count 7.8 × 10² CFU/mL from the truenat location to the linked reference laboratory together with the specimen material may help answer the concerns over contamination or loss of viability of mycobacterium in culture.

The rapidity and accuracy of LPA DST results obtained on truenat-derived DNA from our EP samples, completely concordant with that of LPA DST on culture isolates, are very encouraging. As these findings can have a significant positive bearing on patient management, larger and more comprehensive studies are warranted in multiple settings and under programmatic field conditions to test the expected outcomes in comparison with the reference standard and to further understand the effect of storage and transportation of DNA elute on DST results; the relationship between truenat mycobacterial load and DST outcomes in LPA (DNA elute). If found promising, the practical viability of incorporating DNA elutes for LPA in the national TB program’s diagnostic algorithm may be considered.