Expedited diagnosis of Pediatric Tuberculosis using Truenat MTB-Rif Dx and GeneXpert MTB/RIF

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Expedited diagnosis of Pediatric Tuberculosis using Truenat MTB-Rif Dx and GeneXpert MTB/RIF

https://doi.org/10.21203/rs.3.rs-1624512/v1

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published 28 Apr, 2023

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Rapid, cost-effective, and sensitive diagnostic assays are essential for global tuberculosis (TB) control, especially in high TB burden, resource-limited settings. The current study was designed to evaluate diagnostic accuracy of Truenat MTB-Rif Dx (MolBio) in children less than 18 years of age, with symptoms suggestive of TB. Gastric Aspirate, Induced Sputum, and Broncho-alveolar Lavage samples were subjected simultaneously to AFB-smear, GeneXpert MTB/RIF (GX), liquid culture (MGIT-960) and Truenat MTB-Rif Dx (TN). The index-test results were evaluated against microbiological reference standards (MRS). TN had a sensitivity of 56.1%, specificity of 92.3% against MRS. Sensitivity and specificity of the two tests against liquid culture was 58.3% and 88.3% for TN and 53.6% and 91.9% for GX, respectively. Both GX and TN performed at par. Current study demonstrates that TN can aid early, efficient diagnosis of TB in children.

Pulmonary and intra-thoracic tuberculosis (TB) in children is difficult to diagnose due to the pauci-bacillary nature of disease and varied presentation of involvement of the lungs. More often, younger children present with Progressive primary disease (PPD, lung parenchymal lesion) while older children have mediastinal involvement and primary pulmonary complex (PPC, hilar-lymphadenopathy) on Chest X-ray¹. Issues such as poor/inadequate sample and lower diagnostic yield of laboratory methods further leads to confusion in diagnosis and hence overuse of anti-tubercular treatment for presumptive TB cases.

Detection of bacterial DNA is simpler than detecting live bacteria, which are present only in difficult to reach pulmonary cavities or lymph nodes, with active replication. Recent advancements in molecular diagnostic methods such as Nucleic Acid Amplification tests, e.g. GeneXpert, have brought a sea change to diagnosis of pediatric intra-thoracic TB (ITTB). However, GeneXpert requires a highly sophisticated laboratory with ambient temperature between 15–30°C, stable electricity supply, and adequate storage space for the cartridges (storage at 2–28°C), thereby, limiting its use to district hospital settings. Certain newer NAATs have shown promise in studies done in older patients². The Truenat MTB-RIF Dx assay is a novel point-of-care test, operated on hand held battery device, with minimal operational requirements and potential to be utilized in resource limited settings. The assay involves automated extraction of DNA using bead-based Trueprep device that utilizes a universal cartridge-based system to isolate DNA in 20 minutes, consumables storage 2–40°C. The DNA is then loaded onto the chip-based Truelab micro PCR device and PCR results concluded in 40 minutes. In the absence of optimal diagnostic methods, Microbiological and Clinical Composite Reference standards are often helpful in diagnosis of TB in children as well as in Extra-pulmonary TB. In the present study, consensus case definitions were used to ensure standardized reporting of cases of ITTB for research studies³. Updated definitions now include Xpert MTB/Rif assay (GX) as a diagnostic tool for defining Confirmed TB, in addition to culture as part of microbiological confirmation. Current study was designed to evaluate the diagnostic yield of Truenat MTB-RIF Dx assay (TN) in comparison to the Microbiological Reference Standards (Xpert MTB/RIF assay, MGIT 960 culture and smear), recommended for accurate diagnosis of ITTB in children³.

This multicenter, prospective, cohort study was conducted from August 2018 to August 2019. The study population comprised children less than 18 years of age attending Pediatrics OPD of 2 centers in north India. Children less than 18 years age, presenting with symptoms suggestive of TB, such as cough, fever, loss of weight, were screened for TB by clinical examination, X-ray Chest and enrolled after obtaining written, informed consent from parents or guardian. Gastric Aspirate (GA), Induced Sputum (IS), Bronchoalveolar Lavage (BAL) samples were collected and subjected simultaneously to AFB smear, GX, TN and liquid culture by MGIT-960 (LC). Two samples collected on consecutive days from each patient were pooled and subjected to testing. All conventional procedures for smear, LC and GX were performed following standard Recommendations^4–6. TN was performed as per the manufacturer’s instructions. The TN being a two-step test, Rif chip is tested after initial detection of Mtb⁷. Samples were coded by the statistician at NIMS, ICMR, New Delhi daily. Technicians tested coded samples on Truenat. All tests were conducted in NABL (National Accreditation Board for Testing and Calibration Laboratories) accredited mycobacteriology laboratory. All experiments were performed in accordance with the National ethical guidelines for biomedical and research health involving human participants, 2017 (available at: https://ethics.ncdirindia.org/ICMR_Ethical_Guidelines.aspx).

Microbiological Reference Standards (MRS) included LC, Smear and GX^{1, 8}. Results of the two molecular tests TN and GX were compared to MRS, Smear or Liquid Culture results taken together and to liquid culture alone as well, in order to compare the performance of the two.

Quality Control

It was ensured that, there was no deviation from the set protocol. Laboratory ensured high quality of data and documentation for all methods and is accredited by NABL.

Sample Coding And Blinding For Index Test

Two consecutive days’ samples were pooled and sample aliquots were given code numbers, issued daily for respective samples by NIMS expert. Technician running Truenat test were blinded to the identity of the sample and recorded results against the Code number.

Data Management:

Results from all tests were sent back to NIMS. Expert at NIMS was responsible for decoding and final analysis.

Treatment

The treating physician initiated treatmenton the basis of clinical evidence and MRS. Index test results had no bearing on the decision to initiate treatment.

Statistical analysis

Data were analysed using Stataver 14.1 (SataCorp, College Station, TX). Performance of TN was determined statistically in comparison with GX, LC and Smear using STATA. Data analysis was done per patient. Further both the molecular tests were compared with gold standard culture (with and without smear results).

Ethics

Institute Ethics Committee (IEC), All India Institute of Medical Sciences, New Delhi approved the study protocol (IEC No. IEC- 15/2017). TB treatment decisions were not made based on the result of the TN assay under evaluation, but on the basis of the clinical work-up along with MRS.

Participant Demographics

A total of 612 patients were enrolled during the study period. Out of these, 300 were male while 312 were female patients. The mean age of the patients enrolled was 9.7±3.5 years. Further, the mean age of patients positive by AFB smear was 11.9±2.8,11.1±2.8 by LC, 11.2±3.4 by GX, and 10.9±3.2 years by TN.

From these children, 272 pooled Gastric Aspirates (GA), 337 pooled Induced Sputum samples (IS), and 3 Bronchoalveolar Lavage (BAL) samples were studied. AFB smear, LC, GX and TN were performed in all the samples collected.

Diagnostic Accuracy of Truenat MTB Detection Assays

Of 612 patients, 111 (18.14%) were positive by TN while 47 (7.68%), 84 (13.73%) and 88 (14.38%) were positive by AFB smear, LC, and GX, respectively. Table 1a shows the TN test positivity results in different combinations of smear, LC and GX.

Sensitivity and specificity of the two molecular tests against Smear or LC taken together was 58.3%, 90% for TN and 57.3%, 94.3% for GX. (Table1b). While, sensitivity and specificity of the two against LC alone was 58.3%, 88.3% for TN and 53.6%, 91.9% for GX (Table 1c).

With MRS, TN revealed sensitivity of 56.1%, specificity of 92.3% (Tables 1d). The sensitivity and specificity, positive predictive value and negative predictive value of Truenat in comparison with individual components of MRSare shown in Supplementary Table 1.

Diagnostic Accuracy of Truenat MTB RIF Detection Assays

When comparing detection of Rif resistance between TN and GX (Table 2), few samples were reported Indeterminate by TN.

The current study was designed in a large cohort of children, using state of art methods for diagnosis of presumptive TB.Two nucleic acid detection methodologies Truenat MTB-Rif Dx and GeneXpert MTB/RIF demonstrated comparable performance when evaluated against microbiological reference standards.

MTB culture can detect even low concentrations of organisms. However, the turn-around time for liquid and solid culture systems varies from few days to several weeks and requires sophisticated instrumentation, designated laboratory and specifically trained technical staff. This set-upis not feasible for use in remote areas⁷. There is an urgent need for affordable, easy to use, point-of-care molecular diagnostic assays that augmentthe efforts to treat disease before its spread and irreversible damage to the individual’s health.

The present study is the first prospective, diagnostic comparative analysis of Truenat MTB Rif Dx in pediatric population for the detection of TB and rifampicin resistance. The study found high sensitivity and specificity against microbiological reference standards, thereby confirming its utility as a rapid and accurate assay for diagnosis of presumptive TB in children. The assay results were found equivalent to or better than the Xpert MTB/RIF system, in comparison to microbiological reference standards.

The current study demonstrated similar sensitivity and specificity in children as reported in earlier studies in adults, TN demonstrated sensitivity of 88.3% for detection of MTB in adults², and sensitivity of 87.5% and specificity of 99.5%for detection of Rif resistance⁹. FIND conducted a study in India, Peru, Ethiopia and Papua New Guinea, in peripheral laboratories and primary health centers. Study results confirmed comparable performance with culture and Xpert MTB/RIF Ultra and Xpert MTB/RIF assays at temperatures up to 40^oC and in absence of reliable electricity (with help of battery operated amplification systems)¹⁰.

WHO endorsed the Truenat assays as initial tests toidentify TB and detect Rifampicin resistance following the FIND study. The assays were recommended at the point-of-care in low resource primary healthcare settings¹¹. This assay when utilized as a point-of-care for TB diagnosis in India, demonstrated improvement in linkage-to-care, increase in life expectancy and cost-effectiveness when compared with AFB smear or GX¹².

Strengths of the current study include the study design; few studies have looked at rapid diagnostic modalities of TB in children. The current study was designed to offer a quick but reliable solution for diagnosis of children with presumptive TB. Study design ensured inclusion of a spectrum of clinical presentations. The study definitions were derived using standard recommendations³. The study used state of art methodologies and WHO recommended Nucleic Acid Amplification tests for diagnosis of TB (Xpert MTB/RIF), on a large sample size of patients.

Delays in doing the Rif test, and prolonged storage of extracted DNA leading to degradation of DNA as well as low bacterial load in samples¹⁰ could be responsible for indeterminate results. A recent study reporteddecline in the percentage of invalid results after short training and assay operations after a median of 10 tests¹³.

Current study demonstrated comparable performance of Truenat MTB assays to Xpert MTB/RIF assay, in children. The study results reiterate the potential utility of this cost-effective test in peripheral centers for diagnosis of presumptive TB in children, a difficult to diagnose and treat population. Several studies have demonstrated the use of Xpert MTB/RIF in pediatric population^14–23. With the results of equivalence and comparable performance shown between the two tests in the current study, the other Xpert MTB/RIF study results can be extrapolated. In addition, the manufacturers offer integrated online connectivity, which could facilitate remote monitoring.

Acknowledgements (optional)

The work was supported by grant no. 5/8/5/13/ITRC/2018-ECD-I from the Indian Council of Medical Research, Ministry of Health and Family Welfare, Government of India.

Author contributions

UBS designed the study and wrote the manuscript. MS, KB, AS, NM contributed to Data analysis. JSK, MVVR contributed to statistical analysis with critical inputs from RL. SS, RL and SKK offered clinical care to patients.

Data availability statement (mandatory)

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The author(s) declare no competing interests.

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Table1a. Comparative analysis of test results with Truenat MTB Dx in comparison to Smear, Liquid Culture and Xpert MTB/RIF

Test results	Truenat positive	Truenat negative	Total
Smear + Culture + Xpert +	19	7	26
Smear + Culture + Xpert -	1	1	2
Smear + Culture - Xpert +	9	5	14
Smear + Culture - Xpert -	2	3	5
Smear - Culture + Xpert +	14	5	19
Smear - Culture + Xpert -	15	22	37
Smear - Culture - Xpert +	14	15	29
Smear - Culture - Xpert -	37	443	480
Total	111	501	612

Table 1b. Diagnostic performance of Truenat & GeneXpert with AFB smear or MGIT culture

	AFB smear or MGIT	Both AFB smear and MGIT			AFB smear or MGIT	Both AFB smear and MGIT
Truenat	Pos	Neg	Total	GeneXpert	Pos	Neg	Total
Pos	60	51	111	Pos	59	29	88
Neg	43	458	501	Neg	44	480	524
	103	509	612		103	509	612
Sensitivity 58.3 (48.1, 67.9) Specificity 90 (87, 92.4) Positive predictive value 54.1 (44.3, 63.6) Negative predictive value 91.4 (88.6, 93.7) Positive likelihood ratio 5.81 (4.28, 7.91) Negative likelihood ratio 0.46 (0.36, 0.58)				Sensitivity 57.3 (47.2, 67) Specificity 94.3 (91.9, 96.2) Positive predictive value 67 (56.2, 76.7) Negative predictive value 91.6 (88.9, 93.8) Positive likelihood ratio 10.1 (6.8, 14.9) Negative likelihood ratio 0.45 (0.36, 0.56)

Truenat

TP (n=60): positive by Truenat, AFB smear & MGIT; FP (n=51): positive by Truenat but negative for AFB smear & MGIT; FN (n=43): negative by Truenat but positive for AFB smear & MGIT; TN (n=458): negative by Truenat, AFB smear & MGIT

GeneXpert

TP (n=59): positive by GeneXpert, AFB smear & MGIT; FP (n=29): positive by GeneXpert but negative for AFB smear & MGIT, FN (n=44): negative by GeneXpert but positive for AFB smear & MGIT, TN (n=480): negative by GeneXpert, AFB smear & MGIT

Table 1c. Diagnostic performance of Truenat & GeneXpert with MGIT culture

	MGIT				MGIT
Truenat	Pos	Neg	Total	GeneXpert	Pos	Neg	Total
Pos	49	62	111	Pos	45	43	88
Neg	35	466	501	Neg	39	485	524
	84	528	612		84	528	612
Sensitivity 58.3 (47.1, 69) Specificity 88.3 (85.2, 90.9) Positive predictive value 44.1 (34.7, 53.9) Negative predictive value 93 (90.4, 95.1) Positive likelihood ratio 4.97 (3.7, 6.68) Negative likelihood ratio 0.47 (0.36, 0.6)				Sensitivity 53.6 (42.4, 64.5) Specificity 91.9 (89.2, 94) Positive predictive value 51.1 (40.2, 61.9) Negative predictive value 92.6 (90, 94.7) Positive likelihood ratio 6.58 (4.64, 9.32) Negative likelihood ratio 0.5 (0.4, 0.63)

Truenat

TP (n=49): positive by Truenat & MGIT; FP (n=62): positive by Truenat but negative by MGIT; FN (n=35): negative by Truenat but positive by MGIT; TN (n=466): negative by Truenat & MGIT

GeneXpert

TP (n=45): positive by GeneXpert & MGIT; FP (n=43): positive by GeneXpert but negative by MGIT; FN (n=39): negative by GeneXpert but positive by MGIT; TN (n=485): negative by GeneXpert& MGIT

Table 1d: Diagnostic performance of Truenat with Microbiological reference standard (MRS)

	AFB smear or MGIT or GeneXpert	AFB smear & MGIT & GeneXpert
Truenat	Pos	Neg	Total
Pos	74	37	111
Neg	58	443	501
	132	480	612
Sensitivity 56.1 (47.2, 64.7) Specificity 92.3 (89.5, 94.5) Positive predictive value 66.7 (57.1, 75.3) Negative predictive value 88.4 (85.3, 91.1) Positive likelihood ratio 7.27 (5.15, 10.3) Negative likelihood ratio 0.47 (0.39, 0.57)

Table 2. Comparative analysis of Truenat RIF test results with GeneXpert RIF

TruenatRIF		GeneXpert RIF
	Sensitive	Resistant	Indeterminate	NA	Total
Sensitive	49	0	0	42	91
Resistant	1	2	0	3	6
Indeterminate	3	0	1	10	14
NA	30	2	0	469	501
Total	83	4	1	524	612

Concordance: 521/612 = 85.1%

No competing interests reported.

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You are reading this latest preprint version

Expedited diagnosis of Pediatric Tuberculosis using Truenat MTB-Rif Dx and GeneXpert MTB/RIF

Status:

Journal Publication

Version 1

Abstract

Introduction

Materials & Methods

Quality Control

Sample Coding And Blinding For Index Test

Data Management:

Treatment

Statistical analysis

Ethics

Results

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1