Rapid diagnosis of Mycobacterium tuberculosis with Truenat MTB: a near-care approach.

PLoS ONE 8(1): e51121. doi:10.1371/journal.pone.0051121

Chaitali Nikam1, Manjula Jagannath2, Manoj Mulakkapurath Narayanan2, Vinaya Ramanabhiraman2, Mubin Kazi1, Anjali Shetty1, Camilla Rodrigues1*

1 Department of Microbiology, P. D. Hinduja Hospital and Medical Research Centre,  Mahim, Mumbai, India,

2 bigtecLabs, bigtec Pvt.Ltd, Rajajinagar, Bangalore, India


ABSTRACT

Tuberculosis (TB) causes the highest number of deaths globally, attributable to a curable infectious agent despite the availability of potent anti-TB medication.  Reduction in TB-related morbidity and mortality is impeded by the lack of rapid and cost-effective diagnostic tests that are implementable in resource-limited settings. Over 95% of new TB cases and TB deaths occur in developing countrieswhere smear-microscopy, which detects only 45% of TB infections, remains the most practical and often only test available. Even where infrastructure exists, more sensitive tests are currently time- and cost-prohibitive. Performing a culture can take weeks because of the slow growth rate of TB bacilli (3-6 weeks). Molecular tests such as Nucleic acid amplification Test (NAAT) like polymerase chain reaction (PCR), which are considerably faster than culture, often have a high turnaround time as specimens are often sent to distant laboratories. There has been considerable interest in the efficiency of the PCR platform as this would confer advantages such as reduction in cost of instruments and tests, faster turnaround times and enhancement in the availability and accessibility of PCR tests in resource-poor geographies. With the combined advantages of affordability, simplicity in operations, diagnostic sensitivity and portability, micro-PCR devices are strong candidates for wide scale use among the peripheral laboratories of India and other countries of South-East Asia which account for 50% of the global burden of MTB [1]. The expense involved in PCR testing makes it out of reach of most patients in TB-endemic countries. High risk of transmission of TB makes cost-effective and rapid detection crucial to control of spread of infection.


SUPPLEMENTARY

The study by Chaitali Nikam et.al focused on, evaluation of a novel TB test, Truenat MTB (bigtec Labs, India). The test requires the user to add 5 µl of extracted DNA to a pre-loaded microchip [2] containing room temperature stabilized reagents and PCR run on a handheld battery-operated device, Truelab Uno™ PCR device, that is a fully portable stand-alone thermal cycler [3]. Briefly, the Truelab platform consists of a PDA (personal digital assistant) running the software application, a handheld unit housing the control electronics and optical detection system for real-time monitoring and a microchip with integrated temperature control elements. TheTruenat MTB test involves sputum processing using a battery-operated sample preparation device, Trueprep-MAG™, which extracts nucleic acids by a simple menu driven process using a nanoparticle-based protocol optimized for sputum. The device integrates all operations (heating, fluid mixing, magnet control, step timing) using on a programmed micro-controller, and easy to follow screen instructions, thereby enabling nucleic acid isolation without the need for any additional equipment. (Figure 1) The only equipment required for extraction of DNA is Laminar-air flow for safety of a personnel who is performing the test. The chip-based test has been designed to simplify the process of real-time PCR from ‘sample to result’ so that laboratories with minimal infrastructure can easily perform these tests routinely in their facilities and report PCR results in less than an hour. This is much faster than conventional methods and far more inexpensive as compared to existing methods.

Camilla Rodrigues-fig1

Fig 1: Sample loading on Trueprep-MAG device

 Camilla Rodrigues-fig2

Fig 2: Addition of 5µl of DNA to Truenat MTB chip

 

Existing methods compared are phenotypic (Smear, MGIT960 Culture) and Genotypic (Nested PCR, Real time PCR) methods with which the  results obtained were compared with Composite Reference Standard (CRS). A composite reference standard (CRS) was used to categorise patients. Patients were allocated into the following groups based on a combination of smear status, culture results, clinical treatment and follow-up, and radiology findings.[4]

1. Definite-TB: Smear positive and Culture positive (S+C+) or smear negative, culture positive (S-C+)

2. Probable-TB. Smear positive and culture negative(S+C-) Smear negative and Culture negative(S-C-) but clinical-radiological picture highly indicative of TB. Patients showing a response to anti-TB treatment at follow-up were assigned to this group.

3. Non-TB. Smear negative culture negative (S-C-) Patient was assessed as ‘clinically negative’ when the patient had no previous history of TB infection and no microbiological evidence indicating current infection. Patients were symptomatic (weight loss and prolonged cough) but showed improvement without anti- TB treatment.

Result and Discussion

The study was carried out on total 230 specimens out of these, 4 were detected as non-tuberculous mycobacteria (NTM) by phenotypic and hence were excluded from this study. Of the remaining 226 sputum specimens, 141 were MTB culture positive(C+) and 85 were culture negative(C-). Out of 141 C+,104 patients were on anti-tubercle treatment and 37 were treatment naıve, out of 85 C-, 50 patients were on treatment and 35 were treatment naive. A total of 120 specimens were smear positive (S+).Among the S+ specimens 104 (93.33%) were C+ and 16 S+ specimens failed to grow in culture medium. These S+C- specimens were CRS+. Of the S+, 117 (97.50%) were detected as positive by the IS6110 nested PCR, and 119 (99.16%) were detected as positive by the Truenat MTB test. Of 141 C+ specimens, 135 (95.74%) were detected as positive by the IS6110 nested PCR, and 132 (93.62%) were detected as positive by the Truenat MTB test.Among the S+C+ specimens, both the in-house nested PCR and Truenat MTB detected 99.12% (111/112) of specimens accurately. Among the S-C+ specimens, 75.86% (22/29) were Truenat MTB positive and 82.76% (24/29) were positive by the IS6110 nested PCR protocol. The Truenat MTB results were largely concordant with the inhouse nested PCR results, 196 of 226 specimens showed the same result by either PCR test. Of the 30 discordant results, 16 specimens were MTB positive by nested PCR but not by Truenat. Of this group, 3 specimens were CRS- and treatment naive but consequently false positives. On the other hand, 14 of the 30 were MTB positive by Truenat but not nested by PCR. Of this group, all 14 were CRS+ and on anti-tubercle treatment indicating no false positives. Outcome of the study as shown in Fig.3.

 

Camilla Rodrigues-fig3

Fig. 3  Enlistment and outcome of study.

 

Liquid culture had an average time to positivity (TTP) of 25 days, in-house nested PCR had a TTP of 7 days (additional 7 days if PCR was inhibited) and the Truenat MTB test had a TTP of approximately 1 hour which is very less as compared to phenotypic test.

Discussion

Application of molecular methods in routine for diagnosing in a developing country like India depends on diverse factors like high cost, rapid and accessibility of skilled personnel to perform the test. Although economical and extensively available, microscopy was non-specific in our study population and less sensitive than Nucleic acid amplification Test (NAAT). In present study, the Truenat MTB test was found to have sensitivity of 91.1%(CI: 91.1–94.7) and in- house nested PCR have sensitivity of 90.5% (CI: 85.5–94.3). NAAT demonstrated good specificities in our study as shown in forest plot (Fig. 5) and ROC curve (Fig.4) against smear, culture.

Camilla Rodrigues-fig4

Fig. 4 ROC curves for various techniques evaluated in this study. Performance of molecular tests reporting sensitivity and specificity. The curve is the regression line that summarises the overall diagnostic accuracy. Q* is an index defined by the point on the SROC curve where the sensitivity and specificity are equal, which is the point closest to the top-left corner of the ROC space. SROC: summary receiver operating curve; AUC: area under the curve; SE (AUC): standard error of AUC; SE (Q*): standard error of Q* index.

Camilla Rodrigues-fig5

Fig.5 Forest plot for sensitivity values of microbiological and molecular methods. Forest Plot for sensitivity of smear, Culture, Nested and Truenat molecular methods with pooled sensitivity. Performance of molecular methods studies reporting sensitivity. Point estimates of sensitivity estimates from each study are shown as solid circles. Solid lines represent the 95%CI. CI = confidence interval.

 

Pulmonary TB constitutes the major form of TB in clinical practice. In this study, we evaluated the Truenat MTB test for detection of pulmonary TB in near-care settings.  Based on CRS criteria, PCR assays had a higher sensitivity compared to smear microscopy and culture. In this study, 50 CRS positive TB cases were culture negative. This could be attributed to unequal distribution of mycobacteria in paucibacillary respiratory specimens. Comparison of Truenat MTB results with in-house nested PCR results. The load of bacilli require to obtain a positive culture is 100 viable bacilli, and lower detection limit of conventional PCR is 10 copies and for real-time PCR it is 6 copies. However as our laboratory is a tertiary care laboratory with a referral bias towards non-responders, most of the patients, 154 out of 226 patients were on anti tubercle treatment Therefore in some instances the bacilli may not be viable. Hence these bacilli may not grow in culture but their DNA could be detected using PCR. Various nucleic acid amplification tests (NAAT) have been developed for detection of MTB in sputum. In general, different NAAT tests have been found to have positivity in 95 –100% of smear and culture positive specimens where as the positivity ranges from 40–60% in smear negative paucibacillary pulmonary disease. The positivity of the PCR methods evaluated in this study (99% positivity in S+C+ specimens and.75% in S-C+ specimens for the in-house nested PCR and Truenat MTB) was in accordance with previously observed values. Performance estimates of all tests(sensitivity, specificity, Positive predictive value(PPV), Negative Predictive value (NPV) )using the CRS as a reference standard are presented in Table 1.

Camilla Rodrigues-fig6

Table 1:  Comparison of all methods against CRS as reference standard.

 

All of the NAAT studied showed analytical sensitivities for nucleic acid detection quantitatively equivalent to 1–10 mycobacteria. In terms of sensitivity, assays that detect insertion sequence IS6110 for molecular diagnosis of MTB, such as the one described here, benefit from the fact that IS6110 often presents in high copy numbers. Though culture is inexpensive, the high turn-around time(TAT) is an important barrier to rapid detection. In-house PCR protocols such as the IS6110 nested PCR protocol described here, though sensitive; suffer from false positivity due to inherent pitfalls associated with PCR techniques that require post-amplification analysis (carryover contamination between specimens, reagent contamination due to aerosol-based transmission of amplicon). The TAT can be quite long as specimens need to be batched to increase cost-effectiveness. Additionally, PCR inhibition rates can be high as observed 8.4% in present study, increasing the TAT by a few days, at the same time increasing the overall cost of PCR-based diagnosis. The Truenat MTB test evaluated in this study had a TAT of approximately one hour, enabling rapid detection of MTB DNA. The optimized sputum processing protocol (after standardisation) ensured that PCR inhibitors were removed from the isolated DNA. As only one sample can be process at a time, this reduces the chances of carry over contamination and false positive results.. The disposable, self-contained chip, designed to be a single-use consumable eliminated the possibility of carryover between specimens. The results are displayed on the screen and can be transmitted via GSM/Wi-Fi/ Bluetooth to a central server or printer. The light weight, portable nature of the devices makes them deployable in peripheral laboratories.

The Truenat MTB test not only has good sensitivity and specificity for the diagnosis of TB but also fits the requirements of the resource-limited health care settings. The important benefit of this assay is that it is inexpensive as compared to other NAAT assays, which is an important issue in poorer countries. Health service weaknesses in many countries, including low coverage, user charges, and poor quality of care, highlighting the urgent need for a substantial increase in health sector investment to expand access to preventive and curative health services. Government and non-governmental interventions should also be broadened to encompass measures that reduce the substantial indirect costs associated with diseases such as TB. Large multi-centric studies are required to obtain better estimates of the Truenat MTB performance.

References

  1. Mathema B, Kurepina NE, Bifani PJ, Kreiswirth BN (2006) Molecular Epidemiology of Tuberculosis: Current Insights. Clin Microbiol Rev 19 (4)658–685
  2. Kumar KK, Jayaraman R, Narasimha SK, Radhakrishnan RM, Viswanathan S, Nair CB, Subbarao PV, Jagannath M, Chennakrishnaiah S (2009). PCT Pub. No WO/2009/047805
  3. Kumar KK, Jayaraman R, Narasimha SK, Radhakrishnan RM, Viswanathan S, Nair CB, Subbarao PV, Jagannath M, Chennakrishnaiah S, Mondal S, Venkataraman V (2009). Handheld Micro PCR Device. PCT Pub. No. WO/2009/047804
  4. Minal Deshmukh , Chaitali Nikam, Trupti RagteAnjali Shetty, Camilla Rodrigues ‘Is a composite reference standard (CRS) an alternative to culture in assessment and validation of a single tube nested in-house PCR for TB diagnosis?’: Egyptian journal of chest diseases and tuberculosis (In press , available online 14 oct. 2013)
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