Multi-centric validation of Truenat MTB Rif Dx for Diagnosis of Extrapulmonary Tuberculosis

doi:10.21203/rs.3.rs-4003075/v1

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Multi-centric validation of Truenat MTB Rif Dx for Diagnosis of Extrapulmonary Tuberculosis

https://doi.org/10.21203/rs.3.rs-4003075/v1

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Extra-pulmonary TB (EPTB) is difficult to diagnose due to paucibacillary nature of disease. Current study evaluated accuracy of Truenat MTB-Rif Dx (TN), for detection of Mycobacterium tuberculosis and resistance to rifampicin. Samples were collected from 2103 treatment naive adults with presumptive EPTB, and tested by smear microscopy, liquid culture (LC) (MGIT-960) and GeneXpert MTB/RIF (GX) (Microbiological Reference Standards, MRS). TN results were compared to MRS and Composite Reference Standards (CRS, Microbiology, histopathology, radiology, clinical features prompting decision to treat, response to treatment). TN and GX results were further compared to Clinical Reference Standards (CRS). CRS grouped patients into 551 confirmed, 1096 unconfirmed, and 409 as unlikely TB. TN sensitivity and specificity was 73.7% and 90.4% against GX. Against LC, TN sensitivity and specificity was 62.3% and 84.9%; highest sensitivity (89%) in pus samples and specificity (92%) in CSF was similar to GX. TN sensitivity was better in fluid and biopsy samples, slightly inferior for lymph node aspirates. TN sensitivity for RIF resistance detection was slightly superior to GX. TN detected 169 TB patients initiated on treatment, which were missed by GX. The study establishes TN at par with GX and can be utilized for quick and accurate diagnosis of EPTB.

Biological sciences/Microbiology

Biological sciences/Molecular biology

Extrapulmonary Tuberculosis

Truenat MTB-RIF

GeneXpert MTB/RIF

Composite Reference Standards

Microbiological Reference Standards

Tuberculosis (TB) continues to contribute to high morbidity and mortality worldwide. As per WHO estimates, it accounted for approximately 10 million new cases and 1.5 million deaths in 2020¹. Pulmonary TB remains the primary target for TB control as it accounts for approximately 85% of all TB cases, while extra-pulmonary tuberculosis (EPTB) accounts for about 8–24%². Annual global incidence of EPTB has been increasing over the last decade due to the changing TB control practices, the spread of human immunodeficiency virus (HIV), population growth, and improved detection. In patients with TB and HIV co-infection, the rate of EPTB is reported to be more than 50% of all cases³.

In resource-limited settings, EPTB poses an extensive diagnostic and therapeutic challenge, due to its paucibacillary nature. It is difficult to diagnose with routine diagnostic methods such as culture and acid-fast bacilli (AFB) staining. Furthermore, EPTB has complex clinical presentation, depending on the site and stage of infection and often needs invasive procedure to obtain appropriate specimens, making diagnosis more challenging. Routine cytology and histology cannot differentiate between the diseases caused by TB or non-tubercular mycobacteria, chronic inflammatory conditions, and some other granulomatous conditions. EPTB is thus diagnosed by combination of various diagnostic modalities⁴.

Early diagnosis and detection of drug resistance is crucial for effective treatment and prevention of spread of severe forms of disease in the community. With the advent of Nucleic Acid Amplification tests (NAAT), such as GeneXpert/MTB/RIF and Xpert/RIF/Ultra, the sensitivity of detection of EPTB has improved. MolBio Diagnostics (Goa, India) developed a few NAAT assays (Truenat MTB, Truenat MTB Plus and Truenat MTB-RIF Dx) based on chip-based real-time Polymerase Chain Reaction (PCR) technology that utilizes nrdB gene (Truenat MTB), nrdz and IS6110 gene (Truenat MTB Plus) for the semi-quantitative detection of Mycobacterium tuberculosis (MTB) and rpoB gene (Truenat MTB-RIF Dx) for the detection of rifampicin resistance.

The present study was designed as a blinded, cross sectional validation study, to evaluate the diagnostic performance of Truenat MTB-RIF Dx assay (TN) in comparison to the Microbiological reference standards (MRS) (WHO recommended diagnostics (WRD): smear microscopy (Ziehl Neelsen stain), liquid culture (LC) (MGIT-960) and GeneXpert/MTB/RIF) (GX) (molecular WRD/mWRD) and Composite reference standards (CRS) (microbiological investigations, histopathology, radiology, clinical features prompting decision to treat for TB, response to treatment) in patients with presumptive EPTB.

A multicenter, blinded, prospective study was conducted at three tertiary care centers i.e., All India Institute of Medical Sciences New Delhi, Christian Medical College, Vellore, and Hinduja National Hospital, Mumbai from August 2018 to August 2019. Consecutive patients above 18 years of age with unexplained fever, lethargy, weight loss or failure to thrive with specific extra-pulmonary presentations, such as lymph node enlargement, abscess, features of meningitis, pleural effusion, ascites and symptoms suggestive of presumptive EPTB, were enrolled after obtaining written, informed consent from parents or guardian.

Patients with poor general condition, with high chance of mortality, Immuno-compromised state except HIV infection, co-morbid conditions like malignancy, congenital defects, those who had previously received anti-tubercular drugs were excluded.

Sample collection and processing

Specimens including cerebrospinal fluid (CSF), peritoneal/pleural fluid, lymph node, biopsy sample and pus were collected as appropriate. Samples such as lymph-node aspirates, biopsy and pus were subjected to standard N-acetyl-L-cysteine sodium hydroxide (NALC-NaOH) decontamination and concentration by centrifugation, while samples from sterile sites were processed directly. Samples were aliquoted into two vials and assigned code numbers (generated daily by statistician, JSK). The laboratory was blinded to the clinical presentation and supporting findings of the patients. The processed samples were subjected simultaneously to AFB smear, GX, LC and TN. All conventional procedures for smear, LC and GX were performed following standard recommendations^5–7. TN results were recorded against the respective code number assigned.

Acid- Fast Bacillus (AFB) smear microscopy

Ziehl-Neelsen (ZN) staining was performed on both direct and decontaminated samples. Smears were examined under light microscope for the presence of acid-fast bacilli^5–6.

Liquid culture and liquid culture drug susceptibility testing (DST)

The processed samples were inoculated into MGIT 960 (Becton Dickinson, MD, USA). The tubes were incubated at 37°C and the ones flagged positive were tested for contamination on Mueller Hilton Agar plate followed by confirmation by smear microscopy and TBc identification test (Becton Dickinson, MD, USA). Drug susceptibility testing for rifampicin (RIF) and isoniazid (INH) was performed with the MGIT 960 system, using WHO recommended standard critical concentration of 1 µg/ml for RIF and 0.1 µg/ml for INH.

GeneXpert MTB/RIF (GX)

The GX assay was performed according to manufacturer’s instructions (Cepheid, Sunnyvale, CA). Sample reagent was added in a 2:1 ratio to unprocessed sterile sample or decontaminated sample collected from non-sterile sites, and the tube was manually agitated twice during a 15 min incubation period at room temperature. From the treated sample 2 ml was transferred into the cartridge and loaded onto the GeneXpert instrument. The GeneXpert Dx software (version 4.0, Cepheid) reports results as Mycobacterium complex (MTBC) detected, not detected, invalid, error or no result. The resistance to RIF was reported as RIF resistance detected, not detected or indeterminate. The indeterminate result was reported when the test could not accurately determine whether the bacteria were resistant to RIF. The test was repeated for invalid, error, no result, or indeterminate results for RIF resistance⁷.

Truenat MTB and Truenat MTB-RIF (TN)

Truenat MTB and Truenat MTB-RIF were performed as per manufacturer’s instructions (MolBio Diagnostics, Goa, India). The samples were processed using the Trueprep AUTO sample pre-treatment pack and the DNA was extracted adding two drops of liquefaction buffer to 0.5 ml of sample. The entire content was then transferred to lysis buffer tube and incubated for 5 min. This mixture was then transferred into the cartridge and loaded on to the device to isolate ~ 40 µl of DNA. For amplification 6µl of purified DNA was added to microtube containing lyophilized mastermix and allowed to stand for 30–60 s to get a clear solution. A 6 µl of this clear solution was dispensed into the reaction well of the Truenat MTB chip and loaded to the Truenat Uno Dx instrument. The results were displayed as amplification curve on the analyzer screen on a real time basis during the test run. At the end of the test run the results were reported as MTB detected, not detected or invalid. The test was repeated for invalid results. Indeterminate or error was displayed when the obtained values did not meet the requirements for resistance determination. The test was repeated for sample displaying indeterminate or error⁸.

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^8–9. Results of TN 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 molecular tests, similar comparison was done for GX.

Composite Reference Standards (CRS) included microbiological investigations, histopathology, radiology, clinical features (weight loss, fever, lymph node, night sweats, history of contact and loss of appetite) prompting decision to treat for TB, response to treatment. Patients were grouped as Confirmed/Unconfirmed and Unlikely TB^{4, 10}.

Statistical Analysis

Data were analyzed using Stata version 14.1 (StataCorp, College Station, TX). Performance of TN was determined statistically in comparison with MRS and CRS using STATA. Performance of TN and GX were compared with LC and with Clinical Reference Standards in addition, using STATA. Data analysis was done per patient.

Ethics

Institute Ethics Committee (IEC), All India Institute of Medical Sciences, New Delhi approved the study protocol (IEC No. IEC-5/09.02.2017). TB treatment decisions were not made based on the result of the TN assay under evaluation, but based on the clinical work-up along with MRS.

The three sites namely All India Institute of Medical Sciences New Delhi, Christian Medical College, Vellore, and Hinduja National Hospital, Mumbai contributed 843, 348 & 912 samples respectively. There were 1241 (59%) samples from male patients and remaining 862 (41%) from female patients.

Five types of samples were included in the study, 202 (9.61%) were CSF, 644 (30.62%) were fluids (peritoneal, pleural), 449 (21.35%) were lymph node aspirates, 404 (19.21%) were pus and tissue biopsy each respectively. Of all, 19.6% patients presented with cough, 36% with fever, 25.34% had weight loss in recent months, 7.4% patients had abdominal pain, 6.4% had headache and 21.63% had lymphadenopathy.

Of 2103 samples analyzed 358 samples (17%) were found positive using MGIT culture test, 429 samples (20.4%) were found positive using GX and 486 (23.11%) were positive by TN (Table 1).

Comparison of TN with AFB smear

TN test gave a sensitivity of 89.2% in comparison with smear results and specificity of 79.6%. The positive predictive value of TN was 15.2% and negative predictive value was 99.4% (Table 1).

Comparison of TN with Gold Standard LC for detection of TB

TN when compared with LC as gold standard for EPTB diagnosis gave 62.3% sensitivity and 84.9% specificity (Table 1). The sensitivity, specificity of GX was 67.6% and 89.3% in all sample types collected (Table 3).

The sensitivity and specificity of TN compared to LC in AFB positive specimens was 93.2% and 20.8% while that of GX was 96.6% and 25%. Similarly, the sensitivity and specificity in AFB negative specimens was 56.2% and 85.8% while GX had a sensitivity of 61.9% and specificity of 90.2% (Table 2).

The sensitivity and specificity of TN varied with sample types collected in our study. Pus samples demonstrated highest sensitivity of 89% while lowest sensitivity of 43.8% was detected in fluid samples. Further, highest specificity was seen in CSF (92%) while lowest specificity of 69% was seen in pus samples (Table 3). Similar to results in TN, GX demonstrated highest sensitivity of 89% in pus and specificity of 91.5% in CSF. The lowest sensitivity of GX, 31.3% was seen in fluid samples (43.8% by TN) and specificity of 78.9% in pus samples (69% by TN) (Table 3).

Comparison of TN with GX

TN in comparison with GX gave sensitivity of 73.7% and specificity of 89.8%, positive predictive value of 65% and negative predictive value of 93% (Table 1).

Comparison of TN with Microbiological Reference Standards

Comparing the results of TN with the MRS (smear, culture, GX), the sensitivity was 63% and specificity was 91%, positive predictive value of 71.4% and negative predictive value of 87.4% (Supplementary Table 1).

Table 1

Comparative analysis of test results by TN with WRD
Smear (ZN)						Culture (MGIT)						mWRD (GX)
TN		Pos	Neg	Total	Sensitivity 89·2 (80·4, 94·9) Specificity 79·6 (77·8, 81·3) PPV 15·2 (12·1, 18·7) NPV 99·4 (98·9, 99·7)	TN		Pos	Neg	Total	Sensitivity 62·3 (57, 67·3) Specificity 84·9 (83·2, 86·6) PPV 45·9 (41·4, 50·4) NPV 91·7 (90·2, 93)	TN		Pos	Neg	Total	Sensitivity 73·7 (69·2, 77·8) Specificity 89·8 (88·3, 91·3) PPV 65 (60·6, 69·3) NPV 93 (91·7, 94·2)
	Pos	74	412	486			Pos	223	263	486			Pos	316	170	486
	Neg	9	1608	1617			Neg	135	1482	1617			Neg	113	1504	1617
	Total	83	2020	2103			Total	358	1745	2103			Total	429	1674	2103

Table 2

Diagnostic performance of TN & mWRD in smear positive specimens with MGIT culture
		MGIT				MGIT
AFB smear (+) (n = 83)	TN	Pos	Neg	Total	GX	Pos	Neg	Total
	Pos	55	19	74	Pos	57	18	75
	Neg	4	5	9	Neg	2	6	8
	Total	59	24	83	Total	59	24	83
Sensitivity 93·2 (83·5, 98·1) Specificity 20·8 (7·13, 42·2) PPV 74·3 (62·8, 83·8) NPV 55·6 (21·2, 86·3)					Sensitivity 96·6 (88·3, 99·6) Specificity 25 (9·77, 46·7) PPV 76 (64·7, 85·1) NPV 75 (34·9, 96·8)

Table 3

Comparative analysis of TN and mWRD (GX) with Culture (MGIT) (in all samples and sample type wise)
	Culture (MGIT)					Culture (MGIT)
TN	Pos	Neg	Total	Sensi: 62·3 (57, 67·3) Spec: 84·9 (83·2, 86·6) PPV: 45·9 (41·4, 50·4) NPV: 91·7 (90·2, 93)	GX	Pos	Neg	Total	Sensi: 67·6 (62·5, 72·4) Spec: 89·3 (87·7, 90·7) PPV: 56·4 (51·6, 61·2) NPV: 93·1 (91·7, 94·2)
Pos	223	263	486		Pos	242	187	429
Neg	135	1482	1617		Neg	116	1558	1674
Total	358	1745	2103			358	1745	2103
CSF
	MGIT					MGIT
TN	Detected	Not Detected	Total	Sensi: 66·7 (9·43, 99·2) Spec: 92 (87·3, 95·3) PPV: 11·1 (1·38, 34·7) NPV: 99·5 (97, 100)	Xpert	Detected	Not Detected	Total	Sensi: 66·7 (9·43, 99·2) Spec: 91·5 (86·7, 94·9 PPV: 10·5 (1·3, 33·1) NPV: 99·5 (97, 100)
Detected	2	16	18		Detected	2	17	19
Not Detected	1	183	184		Not Detected	1	182	183
Total	3	199	202		Total	3	199	202
FLUID
	MGIT					MGIT
TN	Detected	Not Detected	Total	Sensi: 43·8 (29·5, 58·8) Spec: 87·4 (84·5, 90) PPV: 21·9 (14·1, 31·5) NPV: 95·1 (92·9, 96·7)	Xpert	Detected	Not Detected	Total	Sensi: 31·3 (18·7, 46·3) Spec: 93·3 (91, 95·2) PPV: 27·3 (16·1, 41) NPV: 94·4 (92·2, 96·1)
Detected	21	75	96		Detected	15	40	55
Not Detected	27	521	548		Not Detected	33	556	589
Total	48	596	644		Total	48	596	644
LYMPH NODE
	MGIT					MGIT
TN	Detected	Not Detected	Total	Sensi: 55·4 (46·7, 63·8) Spec: 84·4 (80, 88·4) PPV: 61·6 (52·5, 70·2) NPV: 80·9 (76·2, 85)	Xpert	Detected	Not Detected	Total	Sensi: 71·9 (63·7, 79·2) Spec: 86·5 (82·1, 90·1) PPV: 70·4 (62·2, 77·8) NPV: 87·3 (83, 90·8)
Detected	77	48	125		Detected	100	42	142
Not Detected	62	262	324		Not Detected	39	268	268
Total	139	310	449		Total	139	310	310
PUS
	MGIT					MGIT
TN	Detected	Not Detected	Total	Sensi: 89 (80·7, 94·6) Spec: 69 (63·6, 74·1) PPV: 45·5 (38, 53·1) NPV: 95·6 (92, 97·9)	Xpert	Detected	Not Detected	Total	Sensi: 89 (80·7, 94·6) Spec: 78 (72·9, 82·4) PPV: 54 (45·7, 62·2) NPV: 96·1 (92·9, 98·1)
Detected	81	97	178		Detected	81	69	150
Not Detected	10	216	226		Not Detected	10	244	254
Total	91	313	404		Total	91	313	404
TISSUE BIOPSY
	MGIT					MGIT
TN	Detected	Not Detected	Total	Sensi: 54·5 (42·8, 65·9) Spec: 91·7 (88·2, 94·5) PPV: 60·9 (48·4, 72·4) NPV: 89·6 (85·8, 92·6)	Xpert	Detected	Not Detected	Total	Sensi: 57·1 (45·4, 68·4) Spec: 94·2 (91·1, 96·5) PPV: 69·8 (57, 80·8) NPV: 90·3 (86·7, 93·2)
Detected	42	27	69		Detected	44	19	63
Not Detected	35	300	335		Not Detected	33	308	341
Total	77	327	404		Total	77	327	404

Comparative performance of TN with GX for detection of RIF resistance

GX detected 360 (17.12%) samples to be sensitive, 55 (2.61%) as resistant and 13 (0.62%) as indeterminate, whereas TN found 234 (11%) sensitive, 39 (1.9%) as resistant and 206 (9.79%) as indeterminate results (Table 4). The resultant concordance between TN and GX was seen in 80.8% of specimens.

The discordant results were seen in 124 samples, highest in lymph node (48) followed by pus (39), tissue biopsy (22), fluid (11) and CSF (4). The discordant results observed was primarily due to low or very low DNA levels in 113 (91.12% of discordant) samples (Supplementary Table 2). Rifampicin resistance by TN was indeterminate in these discordant samples in 111/124 (89.5%) samples detected as positive and 103 (92.8%) of these samples had low/very low DNA quantity. In contrast, GX gave 103/124 samples as sensitive despite 85/103 (82.5%) with low and very low amount of DNA, and 18/124 as resistant, while 13/18 (72.22%) had low/very low DNA. Only 3/124 gave indeterminate results (Supplementary Table 2).

Table 4

Comparative analysis of TN results with mWRD (GX)
TN RIF		GX RIF
	Sensitive	Resistant	Indeterminate	Not detected	Total
Sensitive	165	5*	2*	62	234
Resistant	5*	20	1*	13	39
Indeterminate	98*	13*	5	90	206
Not detected	92	17	5	1510	1624
Total	360	55	13	1675	2103
Concordance: 1700/2103 = 80·8%
*Discordant results observed in a total of 124 samples tested, detailed analysis of the same is provided in Supplementary table no. 1

Comparison of TN with Composite Reference Standards

On further analysis of molecular test results with Composite Reference Standards, Confirmed, Unconfirmed and Unlikely TB, TN gave a sensitivity of 63% and specificity of 91.6% (Table 5). Positive predictive value for TN was 79% and Negative Predictive value of 83.1%.

Table 5

Concordance of Truenat with Composite Reference Standard (Confirmed, Unconfirmed and Unlikely TB)
	Confirmed TB (Clinical+ Microbiological Evidence) (Xpert/MGIT/AFB positive)	Unconfirmed TB (Clinical/ Symptomatic Evidence) (Xpert/MGIT/AFB negative)	Unlikely TB (Neither microbiological nor clinical /symptomatic evidence)	Total
Truenat
Detected	347	92	47	486*
Not Detected	204	1004	409	1617
Total	551	1096	456	2103
Sensitivity 63 (58·8, 67) Specificity 91·6 (89·8, 93·2) Positive predictive value 79 (74·9, 82·8) Negative predictive value 83·1 (80·9, 85·2) Positive likelihood ratio 7·5 (6·11, 9·22) Negative likelihood ratio 0·4 (0·36, 0·45)

Comparison of TN and GX results with Clinical Reference Standards (Composite Reference Standards without Microbiological Reference Standards)

In order to compare the performance of TN and GX, patients were grouped using clinical features, radiology, histopathology, and other accessory investigations but without microbiological investigations as Confirmed, Unconfirmed and Unlikely TB¹⁰. TN gave a sensitivity of 69.6% and specificity of 99.9%, while GX gave a sensitivity of 61.5% and specificity of 100% (Supplementary Table 3 (a). Positive predictive value for TN was 99.8% (GX, 100%) and Negative Predictive value of 84% (GX, 80.6%).

Comparison of TN Positive and GX Negative results with Clinical Reference Standards

TN detected 170 extra samples which were missed by GX, smear and culture. Of these patients, 169/170 were Confirmed TB, while 1 patient was Unconfirmed TB. Of these, 59 (34.9%) samples had high DNA quantity but were missed by GX (Supplementary Table 3 (b). In contrast, samples that were detected by GX but missed by TN (Supplementary Table 3 (c) primarily had low/very low quantity DNA (53.09%). This trend was seen in combined as well as site specific data.

Initial studies evaluating GX gave sensitivity of detection of TB in the range of 25%-95%. The differences in these studies were primarily due to enrollment of different patient populations, different clinical presentations of EPTB, different sample types, processing techniques and comparator gold standards utilized¹¹. WHO recommended the use of GX in EPTB samples¹². However, the yield of GX was low in paucibacillary samples due to limited amount of DNA detected¹³. Several meta-analyses concluded sensitivity from 69–83%^14–16. With the new Xpert Ultra, based on detection of two multicopy gene sequences, improvement in sensitivity was reported¹⁷. Studies using Xpert Ultra on EPTB samples have shown improved performance^18–20.

In the current study, the sensitivity of detection by TN in comparison to LC was 62.3% while specificity was 85%, close to that of GX, 67.6% and 89.3% similar to previous studies in EPTB samples where sensitivity and specificity of TN compared to LC was 65% and 70% respectively²¹. Initial studies evaluating GX had demonstrated similar sensitivity (79%) and specificity (97%) when compared to LC²².

Sensitivity for different extra-pulmonary samples varied, being highest in pus samples, similar by both TN and GX (89%) and lowest in pleural/peritoneal fluids (43.8% by TN, 31.3% by GX). TN gave similar sensitivity to GX for pus samples and CSF, higher sensitivity than GX in fluids and biopsy, and lower sensitivity than GX in lymph node aspirates. In studies using GX Ultra sensitivity from lymph nodes has been reported from 50 to 100%, for CSF 71.4 to 96.4%, and lower sensitivity for pleural fluids 47.6 to 84.2%²³.

Further, sensitivity of RIF resistance detection by TN in comparison to LC DST was 95% while sensitivity of RIF resistance detection by GX was 94.4%. In a previous study published by our group sensitivity of RIF resistance detection was 81.82% with GX²⁴. There was a concordance of 80.8% (1700/2103) in the RIF resistance results by TN and GX. Of 124 samples showing discordant results, substantial number had low/very low amount of DNA. While TN labeled 111/124 as indeterminate RIF resistance, GX reported 103/124 as sensitive despite the low/very low DNA. Low/very low amount of DNA is known to create confusion in detection of RIF resistance due to fallacies in probe binding in GX and failing amplification in TN. Reports caution against inference of resistance in samples with low/very low DNA. Paucibacillary nature of extra-pulmonary samples complicates detection of RIF resistance^25–28.

With respect to Composite Reference Standards (CRS), detection by TN in patients grouped as Confirmed, Unconfirmed and Unlikely TB gave sensitivity of 63% & specificity of 91.6%. Similar studies with GX have shown a sensitivity in similar range. Collection and processing of extra-pulmonary samples offers a challenge. While GX extraction protocol involves decontamination by NaLC-NaOH method followed by centrifugation for all extra-pulmonary samples (except CSF) and loading into the cartridge for GX; TN protocol recommends addition of sample treatment buffer directly to samples (to pellet after centrifugation of CSF, pericardial/peritoneal/pleural fluids) and loading into Autoprep for TN. Paucibacillary samples could possibly have additional loss of bacterial DNA during extraction. [TN/Genexpert kit instructions]

With respect to Clinical Reference Standards (CRS without microbiological investigations), detection by TN in patients grouped as Confirmed, Unconfirmed and Unlikely TB gave sensitivity of 69% & specificity of 99.9%. GX performed slightly inferior with a sensitivity of 61.5% and specificity of 100%. TN detected 170 extra samples (at all three sites), which were missed by GX and other microbiological tests. These patients were all in Confirmed TB group with one patient in Unconfirmed TB group. The amount of DNA in these samples was low/very low in 57% patients, and high in 35%, but was missed by GX. GX detected 113 samples missed by TN (included for TN sensitivity calculation, TN showed sensitivity of 73.7% in comparison to GX). These samples predominantly had low/very low DNA amount, and could have hence been missed by TN^{24, 29–30}.

Further improvement in available NAATs could include simplifying sample processing and inclusion of more genes in the test design to improve sensitivity of TB detection and EPTB diagnosis. Also, inclusion of more gene targets especially for detecting resistance to important drugs in the treatment regimen such as INH and fluoroquinolones, essential for designing treatment regimen for drug resistant TB.

Strengths of the current study include a large sample size included from three tertiary care centers in a high endemic, low resource setting. All patient samples were systematically subjected to WHO recommended diagnostics which were used as gold standard comparator for evaluation of TN. Composite reference standards including clinical suspicion, microbiological investigations, histopathology, radiology and response to treatment used together to further evaluate test results.

The current study establishes TN as a sensitive and specific technique for diagnosis of EPTB. In patients initiated on treatment, TN achieved sensitivity of 69.6% (65.1% for GX) and a higher negative predictive value than GX. Results were similar to WHO approved GX for samples such as pus and CSF, better for samples such as fluids and biopsy, though slightly inferior for lymph node aspirates. In addition, when compared to the LC, sensitivity of TN was slightly superior to GX for RIF resistance detection. TN proved to be instrumental in diagnosing more patients with confirmed TB, where other diagnostic methods failed.

Competing interests

The author(s) declare no competing interests.

Author Contribution

UBS: the conception and design of the work. UBS, CR, DJC: the acquisition and interpretation of data. MS, AS, SK, NM, KJS, KB, MVVR, MK, DS, BT, CVV, DS: analysis of data. AB, AM, VA: Clinical Care of patients. UBS, NM drafting the article and revising it critically for important intellectual content. UBS designed the study and wrote the manuscript. MS, CR, DJC, KB, AS, SK, NM, MK, DS, BT, CVV, DS contributed to Data analysis. JSK, MVVR contributed to statistical analysis with critical inputs from UBS. AB, AM, and VA offered clinical care to patients.

Acknowledgements

This work was supported by the Indian Council of Medical Research, Ministry of Health and Family Welfare, Government of India [grant no. F. No. 5/8/5/14/ITRC/2018/ECD-I].

Data availability statement

(mandatory)

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Multi-centric validation of Truenat MTB Rif Dx for Diagnosis of Extrapulmonary Tuberculosis

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Version 1

Abstract

Introduction

Material & Methods

Sample collection and processing

Acid- Fast Bacillus (AFB) smear microscopy

Liquid culture and liquid culture drug susceptibility testing (DST)

GeneXpert MTB/RIF (GX)

Truenat MTB and Truenat MTB-RIF (TN)

Statistical Analysis

Ethics

Results

Comparison of TN with AFB smear

Comparison of TN with Gold Standard LC for detection of TB

Comparison of TN with GX

Comparison of TN with Microbiological Reference Standards

Comparative performance of TN with GX for detection of RIF resistance

Comparison of TN with Composite Reference Standards

Comparison of TN Positive and GX Negative results with Clinical Reference Standards

Discussion

Declarations

Competing interests

Author Contribution

Acknowledgements

Data availability statement

References

Additional Declarations

Supplementary Files

Status:

Version 1