More sensitive, rapid and affordable diagnostic tools for pulmonary tuberculosis (PTB) are urgently needed. This study aimed to evaluate the performance of EasyNAT MTC2.1 Cartridge (abbreviation: EasyNAT) (Ustar Biotechnologies, China), a new isothermal amplification method with a response time of fewer than two hours that requires a few manual steps to process sputum.
Sputum samples from 249 patients with suspected PTB underwent smear, culture, Xpert MTB/RIF (Cepheid, USA), and EasyNAT assay tests. Of the 169 PTB patients, EasyNAT detected more PTB patients than Xpert (72.19% vs. 61.54%, P < 0.05, χ2 = 4.326). Both the Xpert assay and the EasyNAT assay successfully detected almost all culture-positive sputum, but EasyNAT yielded more positive results among smear-negative, culture-negative PTB cases (44.59% (33/74) vs. 97 % (17/74), P < 0.01, χ2 = 7.732).
Although the specificity of EasyNAT was lower compared to Xpert [95.00% (76/80) vs. 98.75% (79/80)], the difference was not significant (P = 0.363, χ2 = 0.826). EasyNAT could be used as an initial test for the diagnosis of PTB due to its simplicity, fast turnaround time, high sensitivity, and low cost.
Keywords: Tuberculosis, diagnosis, Ustar EasyNAT MTC assay, Xpert MTB/RIF, point-of-care testing
Materials and methods
- Study design
From January 2019 to January 2020, sputum samples with a minimum volume of 5 mL were prospectively and continuously collected from patients with suspected TB. All of these patients had symptoms suggestive of TB, including but not limited to prolonged fever, persistent cough, night sweats, and weight loss. In addition, an abnormal radiological chest image was also presented.
Sputum was tested for smear, Lowenstein-Jensen (LJ) solid culture medium, Mycobacterial Growth Indicator Tube (MGIT) 960 culture, Xpert MTB/RIF assay (Cepheid, USA), and assay test. EasyNAT MTC. The study was approved by the Ethics Committee of the Beijing Chest Hospital, Capital Medical University. Since all the samples used were leftover specimens from routine clinical examinations, the written informed consent of the patients was waived.
- Diagnosis and categorization of patients
Enrolled patients were diagnosed according to the Composite Reference Standard (CRS), which comprises clinical findings, laboratory results, radiological images, and follow-up data. Patient categories were defined according to the following criteria: (1) Confirmed TB: smear positive and/or culture positive, ie, MTB was identified.
Some patients initially had negative smear and culture results in this study, but their subsequent examinations produced positive bacterial evidence. Therefore, these patients were also classified as patients with confirmed PTB but were analyzed separately. (2) Probable TB: bacteriological evidence of TB was not acquired and the diagnosis of PTB was based only on symptoms, radiological images, response to treatment and follow-up data. (3) No TB: cancer or other diseases diagnosed by histopathological examination or other tests.
- Smears and culture
A direct smear was prepared and stained with auramine, and then examined with light-emitting diode microscopy. A 2 mL sample was decontaminated with 2–4 mL of 2% N-acetyl-L-cysteine-sodium hydroxide (BBL MycoPrep; Becton Dickinson, Sparks, MD) for 20 min, neutralized with sterile phosphate-buffered saline ( PBS; pH 6.8) to a final volume of 45 mL and then centrifuged at 3000 rpm for 15 min at 4°C.
The pellet was resuspended in 1.5 mL of PBS; 0.5 mL was inoculated into the MGIT 960 system (Becton, Dickinson and Company, USA) and 0.5 mL into the LJ solid medium. All positive cultures were tested with MPT64 antigen to confirm the presence of MTB (HANGZHOU GENESIS BIO DETECTION AND BIOCONTROL CO., LTD, China).
- Xpert MTB/RIF test
The Xpert assay was performed according to the manufacturer’s instructions. 1 to 2 mL raw sputum samples were mixed with 2 volumes of Xpert Specimen Processing Reagent, vortexed for at least 10 s, and then incubated at room temperature for 10 min. The samples were then mixed by vortexing for 10 s and incubated at room temperature for another 5 min. A total of 2 mL of the mix was transferred to the Xpert cartridge and loaded onto the GeneXpert instrument. Next, the automatic detection procedure was executed.
- EasyNAT MTC Assay
Samples of 1–2 mL were mixed with 2 or 4 volumes of 4% sodium hydroxide solution depending on sputum viscosity, then vortexed for 30 s and allowed to stand for 15 min at room temperature until clear. completely liquefied. One mL of liquefied sputum was transferred to a 1.5 mL Eppendorf tube and centrifuged at 12,000 rpm for 3 min.
The pellet was suspended with DNA extraction fluid premixed with the internal control, then transferred to the reaction cartridge and placed in the isothermal equipment. DNA extraction, DNA purification, target gene amplification and target gene detection can be performed on the cartridge within 90 min.