Application of real-time simultaneous amplification and testing method to accurately and rapidly detect extra-pulmonary tuberculosis

Tuberculosis (TB) is a chronic infectious disease that endangers human health for a long time. Its clinical manifestations are extremely diverse, with 80% of TB infections occurring in the lungs and other sites like cervical lymphadenitis, meninges, peritoneum, intestines, skin, and bones [1]. According to the WHO Global Tuberculosis Report 2015 32,000 cases of extra-pulmonary tuberculosis (EPTB) were diagnosed in nearly 530,000 new cases in China in 2014 [2]. In 2018 there were an estimated 10.0 million people fell with TB and 1.2 million death [3]. It is difficult to diagnose EPTB due to its extensive lesions and complicated clinical manifestations.

Rapid and accurate laboratory testing technology plays an important role in assisting the early diagnosis of EPTB in clinical practice. Simultaneous Amplification and Testing (SAT) is one of the molecular biological detection methods used by the tuberculosis laboratory in China in recent years. It is mainly used to detect MTB in sputum to assist diagnosis [4‐7]. However, SAT has rarely been used to test EPTB specimens and therefore, its sensitivity and specificity for EPTB diagnosis are still not clear. This study aimed to evaluate the clinical accuracy of SAT in the diagnosis of EPTB by comparing sensitivity, specificity, positive predictive value and negative predictive value of both SAT and culture methods in detecting EPTB specimens.

Simultaneous Amplification and Testing (SAT) is a nucleic acid detection technology. The nucleic acid isothermal amplification and real-time fluorescence detection are combined in this technology. The principle is that MTB 16S rRNA was reverse transcribed using Moloney murine leukemia virus (M-MLV) reverse transcriptase in order to generate a 170-bp DNA fragment using a pair of specific primers incorporating the T7 promoter sequence in the sense primer. The DNA was transcribed to RNA by using T7 RNA polymerase to undergo successive cycles of amplification. An internal labeled probe was included and released a fluorescence signal when hybridized with the target RNA. The dynamic measurements of the real-time amplification fluorescence signal were detected using a real-time PCR instrument. This technology has been widely used in the clinical detection of MTB in sputum for the early diagnosis of tuberculosis, but the clinical value for the detection of EPTB is rarely reported.

Culture of M. tuberculosis is the gold standard for diagnosis of active MTB from clinical samples. In this study the Roche method, Bactec MGIT 960 liquid culture method and SAT method were used simultaneously to detect MTB of EPTB specimens. Using the culture methods as the gold standard, the sensitivity and specificity of TB-SAT detection were 83.6 and 79.4%, respectively, and the agreement of the two methods was 80.5%. Most literature reports SAT applied to the detection of MTB sensitivity in sputum in 21.2–75.8% and specificity in 94.73–100% [4‐7, 9]. Fan Lin et al. believed that the differences are related to the treatment of sputum specimens, transport and focal pulmonary tuberculosis, not with the method itself. Using clinical diagnosis as the gold standard, the sensitivity and specificity of the TB-SAT detection were 41.6 and 100% in our study, respectively, which was slightly higher than the culture methods whose sensitivity and specificity were 29.3 and 98.0%, respectively. This was mainly due to the positive results of SAT detection could exclude single infection of non-tuberculosis mycobacterium (NTM). However, SAT could not detect mixed infection of MTB and NTM in the same sample. Additionally, the detection target of SAT-TB method is rRNA. RNA products are much more labile outside the reaction tube than DNA amplification products. Base on the higher specificity of SAT test that can generally be completed within 2 h, when both the SAT and sputum smears are positive, it is most likely for the patient to be infected with the MTB. Although, in this study, the negative predictive value of SAT was low, suggesting that clinicians cannot exclude tuberculosis based on the negative SAT test result and still need to use other test results to confirm the diagnosis.

Roche culture method is a traditional and classical laboratory method in the detection of mycobacteria and has long been considered as the gold standard for the diagnosis of tuberculosis, and its test results are usually used as the gold standard for evaluating new MTB detection reagents. Taking into account the mycobacterium vitality and Roche’s nutrient status of roche medium, this study used Roche culture method and Bactec MGIT 960 liquid culture method to detect specimens at the same time in order to improve the sensitivity of the culture method detection results. Bactec MGIT 960 liquid culture method was contrast with SAT test and Roche culture method was used identified the samples with discordant results between the Bactec MGIT 960 liquid culture and SAT-TB methods. The real-time fluorescent PCR method has high sensitivity and specificity for detection of MTB nucleic acid. When the results of SAT detection and culture method are inconsistent, another real-time fluorescent PCR detection reagent is used for evaluation. There was also a large cohort study to further analyze the specificity of the SAT detection method [10]. When the test results are inconsistent, a repeat test of SAT is performed with a detection sensitivity of 75.8%. In this study, 94 specimens with inconsistent TB-SAT and culture methods were tested for MTB identification. The agreement between TB-SAT and real-time fluorescence PCR was 92.6% (87/94). In order to confirm the consistency among the methods, 21 strains of TB-SAT-negative and culture-positive strains were tested for mycobacterial species identification in this study. The results showed that only 5 cases were NTM and the other 16 cases were MTB accounted for 76.2% (16/21), which means that patients with SAT-negative and culture-positive patients should not be considered clinically infected with MTB. Three cases with negative TB-SAT test, positive cultures results and positive MTB assays were analyzed for possible reasons: impurities in the samples affecting TB-SAT amplification reaction, or special components in non-sputum samples (such as pus, tissue, etc.) leads to uneven distribution of target in the sample, or less bacteria in the sample and uneven distribution of template in the lysate, etc. 73 cases with negative culture method and TB-SAT positive samples were identified by PCR. The results showed that 70 cases of TB-SAT and PCR were positive, accounting for 95.9% (70/73). The other 3 cases were culture negative and TB-SAT positive. Samples that were negative by PCR were not confirmed by further tests. This was a deficiency of this study. In addition SAT detection was also an asset in early detection and management of PTB suspects, especially in those patients who are smear negative or sputum scarce and low cost and rapidity [9, 10].

As same as all detection methods, there are some limitations of SAT for detection of EPTB. The first is the pretreatment of EPTB sample that is different from PTB. Some EPTB samples need more time to be processed such as bone, lymph node and so on. The second is RNA degradation. The principle of SAT is that MTB 16S rRNA was reverse transcribed using Moloney murine leukemia virus (M-MLV) reverse transcriptase in order to generate a 170-bp DNA fragment using a pair of specific primers incorporating the T7 promoter sequence in the sense primer. The DNA was transcribed to RNA by using T7 RNA polymerase to undergo successive cycles of amplification. If large RNA degraded quickly, the result may not be right. The Third is the number of live MTB in the EPTB sample. Enough live MTB can produce a large of RNA for detection. The lack of live MTB may affect detection results. In view of the above three limitations EPTB sample should be processed quickly and correctly in order to improve the results of SAT detection.

In summary, SAT has the advantages of stable reaction, high sensitivity and specificity, simple reaction conditions, simple operation, no temperature cycling, high amplification efficiency, and short reaction time; RNA amplification products and templates are both RNA and RNA are easily degraded and laboratory contamination can be greatly reduced. SAT-TB detection is a simple, rapid, sensitive and specific tuberculosis detection method. It can be used clinically to assist in the diagnosis of EPTB and at the same time to some extent increase the positive rate of EPTB patients. SAT-TB is a higher clinical diagnosis value for EPTB in clinical microbiology laboratories.

SAT test is a simple and rapid test with high specificity for the detection of EPTB. SAT-TB is a higher clinical diagnosis value for EPTB in clinical microbiology laboratories.