Comparative Study of a Real-Time PCR Assay Targeting senX3-regX3 versus Other Molecular Strategies Commonly Used in the Diagnosis of Tuberculosis

Abstract

BackgroundNucleic acid amplification tests are increasingly used for the rapid diagnosis of tuberculosis. We undertook a comparative study of the efficiency and diagnostic yield of a real-time PCR senX3-regX3 based assay versus the classical IS6110 target and the new commercial methods.MethodsThis single-blind prospective comparative study included 145 consecutive samples: 76 from patients with culture-confirmed tuberculosis (86.8% pulmonary and 13.2% extrapulmonary tuberculosis: 48.7% smear-positive and 51.3% smear-negative) and 69 control samples (24 from patients diagnosed with non-tuberculous mycobacteria infections and 45 from patients with suspected tuberculosis which was eventually ruled out). All samples were tested by two CE-marked assays (Xpert®MTB/RIF and AnyplexTM plus MTB/NTM) and two in-house assays targeting senX3-regX3 and the IS6110 gene.ResultsThe detection limit ranged from 1.00E+01 fg for Anyplex, senX3-regX3 and IS6110 to 1.00E+04 fg for Xpert. All three Xpert, senX3-regX3 and IS6110 assays detected all 37 smear-positive cases. Conversely, Anyplex was positive in 34 (91.9%) smear-positive cases. In patients with smear-negative tuberculosis, differences were observed between the assays; Xpert detected 22 (56.41%) of the 39 smear-negative samples, Anyplex 24 (61.53%), senX3-regX3 28 (71.79%) and IS6110 35 (89.74%). Xpert and senX3-regX3 were negative in all control samples; however, the false positive rate was 8.7% and 13% for Anyplex and IS6110, respectively. The overall sensitivity was 77.6%, 85.7%, 77.3% and 94.7% and the specificity was 100%, 100%, 90.8% and 87.0% for the Xpert, senX3-regX3, Anyplex and IS6110 assays, respectively.ConclusionReal-time PCR assays targeting IS6110 lack the desired specificity. The Xpert MTB/RIF and in-house senX3-regX3 assays are both sensitive and specific for the detection of MTBC in both pulmonary and extrapulmonary samples. Therefore, the real time PCR senX3-regX3 based assay could be a useful and complementary tool in the diagnosis of tuberculosis.

Diagnostic accuracy of in-house real-time PCR assay for Mycobacterium tuberculosis: a systematic review and meta-analysis

“… MGIT, LJ,7H11 sp,ba,bal.,ti In,ab,pf,ti 89/23 30 4 7 71 Rao 2016 [ 30 ] Cross-sectional India 100 200 44/56 16sRNA ? MGIT sp – 100/0 44 2 0 54 Rozales 2014 [ 31 ] Cross-sectional Brazil 447 447 42/405 IS6110 ZN 7H9,MGIT sp,bal – 124/0 41 7 1 75 Sanjuan-Jimenez 2015 [ 32 ] Case-control Spain 153 145 76/69 senX3-regX3 ZN LJ,MGIT sp,ba,bal pf,In,ur,csf,ar 125/20 65 0 11 69 Sanjuan-Jimenez 2015 [ 32 ] Case-control Spain 153 145 76/69 IS6110 ZN LJ,MGIT sp,ba,bal pf,In,ur,csf,ar 125/20 72 9 4 60 Acid-fast stain: ZN, Ziehl-Neelsen; Culture: MGIT, Mycobacteria growth indicator tube; LJ, Löwenstein-Jensen; 7H9, Middlebrook 7H9 Broth. Respiratory specimen: sp., sputum; ba, broncheal/tracheal aspirate; bal., bronchialalveolar lavage; ti, tissue specimen.…”

“…Medical resources are limited in the majority of these countries [ 1 ]. Although recent studies have revealed that RT-PCR assays have good diagnostic performance for TB, there are unignorable discrepancies between their results [ 10 , 20 – 32 ]. Moreover, none of the researchers could demonstrate precise diagnostic accuracy due to their limited statistical power.…”