Evaluation of the hyplex® TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples

<p>Abstract</p> <p>Background</p> <p>Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen <it>Mycobacterium tuberculosis </it>complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex^®TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of <it>M. tuberculosis </it>directly in clinical samples.</p> <p>Results</p> <p>A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex^®TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex^®TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively.</p> <p>Conclusions</p> <p>The hyplex^®TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of <it>M. tuberculosis </it>in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex^®TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens.</p

A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB

Rapid diagnosis of tuberculosis (TB) and antibiotic resistances are imperative to initiate effective treatment and to stop transmission of the disease. A new generation of more sensitive, automated molecular TB diagnostic tests has been recently launched giving microbiologists more choice between several assays with the potential to detect resistance markers for rifampicin and isoniazid. In this study, we determined analytical sensitivities as 95% limits of detection (LoD(95)) for Xpert MTB/Rif Ultra (XP-Ultra) and BD-MAX MDR-TB (BD-MAX) as two representatives of the new test generation, in comparison to the conventional FluoroType MTB (FT-MTB). Test matrices used were physiological saline solution, human and a mucin-based artificial sputum (MUCAS) each spiked with Mycobacterium tuberculosis in declining culture- and qPCR-controlled concentrations. With BD-MAX, XP-Ultra, and FTMTB, we measured LoD(95)(TB) values of 2.1 cfu/ml (CI95%: 0.9-23.3), 3.1 cfu/ml (CI95%: 1.288.9), and 52.1 cfu/ml (CI95%: 16.7-664.4) in human sputum;of 6.3 cfu/ml (CI95%: 2.931.8), 1.5 cfu/ml (CI95%: 0.7-5.0), and 30.4 cfu/ml (CI95%: 17.4-60.7) in MUCAS;and of 2.3 cfu/ml (CI95%: 1.1-12.0), 11.5 cfu/ml (CI95%: 5.6-47.3), and 129.1 cfu/ml (CI95%: 82.8-273.8) in saline solution, respectively. LoD(95) of resistance markers were 9 to 48 times higher compared to LoD(95)(TB). BD-MAX and XP-Ultra have an equal and significantly increased analytical sensitivity compared to conventional tests. MUCAS resembled human sputum, while both yielded significantly different results than normal saline. MUCAS proved to be suitable for quality control of PCR assays for TB diagnostics

Multi-centre evaluation of the speed-oligo Mycobacteria assay for differentiation of Mycobacterium spp. in clinical isolates

<p>Abstract</p> <p>Background</p> <p>A new DNA line probe assay (Speed-oligo Mycobacteria, Vircell) has been launched for rapid differentiation of <it>Mycobacterium </it>spp. from cultures. Compared to other line-probe assays, Speed-oligo Mycobacteria covers a relatively limited spectrum of species but uses a simpler and faster dip-stick technique. The present multi-centre, multi-country study aimed at evaluating the utility and usability of Speed-oligo Mycobacteria in routine mycobacteriology diagnostics. Results from Speed-oligo Myobacteria were compared to those from Genotype CM (HAIN lifescience, Nehren, Germany), another line-probe assay.</p> <p>Methods</p> <p>Speed-oligo Mycobacteria assay was performed in three main steps: 1) DNA extraction from cultured material 2) PCR amplification of the target gene and an internal control and 3) hybridization of the PCR products to specific probes by means of a dip-stick.</p> <p>Results</p> <p>Two hundred forty-two clinical isolates were recovered from consecutive positive mycobacterial cultures at two German (IML Gauting, Bioscientia Ingelheim), one Czech (KLINLAB Prague), and at a Sudanese (Khartoum) laboratory. All <it>Mycobacterium </it>species covered by the assay were reliably recognized. The rate of false positive results was 1.2% and concerned only the species <it>M. marinum </it>and <it>M. peregrinum</it>. The identification rate, i.e. the proportion of isolates which was correctly differentiated to the level of species or complex by the assay, differed significantly among laboratories being 94.9%, 90.7%, and 75.0% at the study sites IML Gauting, KLINLAB Prague and Bioscientia Ingelheim, respectively. This difference was caused by different spectra of NTM species encountered by the laboratory centres in daily routine diagnostics.</p> <p>Conclusions</p> <p>Speed-oligo Mycobacteria assay was proved a rapid and easy-to-perform alternative to conventional line-probe assays. The assay showed excellent sensitivity with regard to identification of genus <it>Mycobacterium </it>and species/complexes covered by the test. However, due to its relatively limited spectrum of taxa, a varying proportion of NTM may not be identified by the assay in daily diagnostics demanding further analyses. The only significant shortcoming in terms of specificity was the misidentification of the clinically relevant species <it>M. marinum</it>.</p

Emergence of Low-level Delamanid and Bedaquiline Resistance During Extremely Drug-resistant Tuberculosis Treatment.

Two new drugs, delamanid and bedaquiline, have recently been approved for treatment of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis. Here, we report a case of clofazimine, bedaquiline, and low-level delamanid resistances acquired during treatment of a patient with XDR tuberculosis