Multicentric validation of indigenous molecular test Truenat™ MTB for detection of Mycobacterium tuberculosis in sputum samples from presumptive pulmonary tuberculosis patients in comparison with reference standards.

BACKGROUND & OBJECTIVES: : Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator. METHODS: : The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers. RESULTS: : The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture. INTERPRETATION & CONCLUSIONS: : Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.

Validation of an indigenous assay for rapid molecular detection of rifampicin resistance in presumptive multidrug-resistant pulmonary tuberculosis patients.

BACKGROUND & OBJECTIVES: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. METHODS: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. RESULTS: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. INTERPRETATION & CONCLUSIONS: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.

mRNA and DNA PCR tests in cutaneous tuberculosis.

BACKGROUND: The microbiologic diagnosis of cutaneous tuberculosis is difficult because most lesions harbor only a small number of mycobacteria that cannot usually be detected by staining for the organism or by culture. Nucleic acid amplification tests based on the polymerase chain reaction (PCR) are potentially useful in this situation. AIMS: To evaluate the utility of mRNA PCR and DNA PCR in the diagnosis of cutaneous tuberculosis. METHODS: Biopsies from 28 cases of cutaneous tuberculosis and 19 controls with other diseases were subjected to microbiologic tests including direct smears for mycobacteria, culture and both mRNA PCR and DNA PCR. The laboratory was blinded to the clinical diagnosis. RESULTS: None of the patients or controls showed a positive reaction on mRNA PCR test. Seven of 28 cases and 5 out of 19 controls showed a positive result on DNA PCR test yielding a sensitivity of 25% and a specificity of 73.7%. CONCLUSION: The results of PCR tests in cutaneous tuberculosis should be interpreted in the light of clinical and histopathological findings.

Primary tuberculosis of glans penis after intravesical Bacillus Calmette Guerin immunotherapy.

A 55-year-old male with carcinoma in situ of urinary bladder was treated with weekly intravesical injections of Bacillus Calmette Guerin (BCG) vaccine. Three days after the sixth injection, he developed low grade fever and multiple grouped punched out, 2-3 mm ulcers around meatus and corona glandis. In addition, multiple, firm, indurated, nontender papules and few deeper nodules were present on the proximal part of glans penis, along with bilateral enlarged, matted and nontender inguinal lymph nodes. There was no history suggestive of sexually transmitted diseases and high risk behavior. Chest X-ray was within normal limits, and Mantoux, Venereal Disease Research Laboratory (VDRL) and HIV antibody tests were negative. The biopsy from the penile ulcer revealed epithelioid cell granuloma with Langhans giant cells. Fine needle aspiration cytology from the lymph node also revealed epithelioid cell granuloma and acid fast bacilli on Ziehl Neelsen's stain. The tissue biopsy grew Mycobacterium tuberculosis. The BCG immunotherapy was stopped and patient was treated with four drug antitubercular therapy with isoniazid, rifampicin, ethambutol, and pyrazinamide in standard daily doses along with pyridoxine. The edema resolved and the ulcers started healing within 2 weeks, and at 6 weeks after starting antitubercular therapy almost complete healing occurred. To the best of our knowledge, we describe the first case of an Indian patient with BCG induced primary tuberculosis of penis after immunotherapy for carcinoma urinary bladder and review the previously described cases to increase awareness of this condition in dermatologists and venereologists.