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Abstract
Tuberculosis (TB) causes a heavy health burden worldwide, especially in developing countries. The need for the rapid and accurate diagnosis of TB has not been satisfied, especially for extra-pulmonary specimens or specimens with acid fast stain (AFS)-negative condition. Development and validation of a novel, sensitive and specific assay for diagnosing TB is essential. We developed IS4 primer/probe based on insertion sequence 6110 (IS6110). A qPCR assay was designed for detecting a specific region in IS6110 by BLAST. The IS4 primer/probe concentration, qPCR efficiency and various of PCR additives were evaluated and optimized. Thirty-four species of commonly isolated microorganisms were used for evaluating the analytical specificity. Moreover, 130 clinical specimens were collected for evaluating the performance versus Cobas TaqMan MTB (CTM) assay kit and culture. The amplification efficiencies of IS4 were 99.61% and 102.61% without and with internal control DNA (Bacteriophage Lambda), respectively. Dimethyl sulfoxide outperformed glycerol or BSA for eliciting the most effective amplification and the lowest limit of detection. In evaluating the clinical performance, various specimen types were collected. IS4 demonstrated a high degree of agreement (kappa = 0.71) with CTM. The clinical sensitivity and specificity of IS4 and CTM were 92.11% (35/38), 82.61% (76/92), 84.21% (32/38) and 95.65% (88/92), respectively. The clinical sensitivity and specificity of IS4 were similar for both pulmonary [92.00% (23/25) and 76.92% (30/39), respectively] and extrapulmonary [92.31% (12/13) and 86.79% (46/53), respectively] specimens. Among AFS-negative cases, the clinical sensitivity and specificity remained 90.48% (19/21) and 83.91% (73/87), respectively, with culture as the gold standard. We concluded that IS4, a new primer/probe pair for TaqMan based qPCR assay, was developed, optimized, and validated for the sensitive and specific detection of TB among various specimen types. The performance was not compromised under AFS-negative conditions.
Highlights
Tuberculosis (TB) infects approximately 10.4 million people and causes 1.4 million deaths annually[1]
It has been reported that insertion sequence 6110 (IS6110) could be widely used for TB diagnosis in clinical specimens; previous studies have established the validity of the detection rate and sensitivity of MTB complex (MTBC) targeting IS6110, showing that it was significantly higher than culturing MTB for a long time period[15,16,17,18]
nucleic acid amplification tests (NAATs) are recommended by the World Health Organization (WHO) as initial tools for cases suspected to have TB
Summary
Tuberculosis (TB) infects approximately 10.4 million people and causes 1.4 million deaths annually[1]. It has been reported that IS6110 could be widely used for TB diagnosis in clinical specimens; previous studies have established the validity of the detection rate and sensitivity of MTBC targeting IS6110, showing that it was significantly higher than culturing MTB for a long time period[15,16,17,18] Both commercial and laboratory developed test (LDT) NAATs have been developed based on these target regions. In addition to CTM, the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is another commercialized NAATs, which approved by World Health Organization for rapid TB diagnosis, allows completely automated nuclei acids preparation, amplification, and detection rifampin resistance of M. tuberculosis by quantitative PCR with a disposable single test cartridge in the meantime One study indicated it has attained 100% sensitivity and 67% specificity with AFS-positives cases; whereas those values were 40% and 99%, respectively with AFS-negative cases[21]. Re-addressing the issue of MTBC NAATs and developing an efficient LDT NAAT is essential
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