Xpert® MTB/RIF assay on formalin-fixed paraffin-embedded tissues in the diagnosis of extrapulmonary tuberculosis

Abstract

BackgroundDiagnosis of extrapulmonary tuberculosis continues to be a challenge due to the complexity of the causative organism and the wide array of pathologic features seen in this infection. Xpert MTB/RIF can be used on fresh or frozen tissue specimens for diagnosis of tuberculosis with good results. However, there is little data on its use with formalin-fixed paraffin-embedded (FFPE) tissues.ObjectivesThe aim of this study was to demonstrate the potential utility of Xpert MTB/RIF and to compare its performance to Ziehl-Neelsen staining for the detection of Mycobacterium tuberculosis from FFPE tissues using histological features from haematoxylin and eosin staining as the gold standard.MethodsEighty randomly selected archival FFPE tissues exhibiting histological features of tuberculosis were included in the study. After deparaffinisation and lysis, all the tissue specimens were subjected to the Xpert® MTB/RIF assay. The outcome measures were proportions of positively identified cases by each test.ResultsUsing histology as the gold standard, the sensitivity of Ziehl-Neelsen staining was 20.3% (95% confidence interval: 12% – 30.8%), and the sensitivity of the Xpert® MTB/RIF assay was 53.2% (95% confidence interval: 41.6% – 64.9%); the difference was statistically significant (p = 0.002). None of the cases tested positive for rifampicin resistance.ConclusionWith prior deparaffinisation and lysis, FFPE tissues are amenable to testing by Xpert® MTB/RIF assay. A validation study to determine the clinical utility, analytical optimisation and cost implications of this assay for FFPE tissues is recommended.

A novel and simple heat-based method eliminates the highly detrimental effect of xylene deparaffinization on acid-fast stains

“…For a definitive diagnosis the demonstration of acid-fast bacteria is necessary, usually done by ZN staining, which is well known to be often negative. 3,5,6 Interestingly fluorescent AFS, such as AuO, which are superior to ZN and have been endorsed by the World Health Organization (WHO) for microbiology diagnosis are not widely used in histological diagnosis. 25 The results of this study show that mycobacteria in tissues are highly fluorescent after staining with standard AuO with acceptable background fluorescence in tissues (figure 1).…”

“…However, while histology often reveals TB-like tissue lesions, such as granulomas, AFS stained sections, needed for a definitive diagnosis, are often negative for mycobacteria. 3,5,6 Using AFS on deparaffinized histology sections is well-known for its poor yield which is attributed to the low number of mycobacteria present in the tissue. 3,5,7–9 However, could it be that histologic processing and the staining procedures themselves cause the unreliable detection of mycobacteria?…”

Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study

“…Sensitivities of Xpert ® MTB/RIF and its ultra version reported in fresh non-FFPE clinical samples, so far, are always higher than 60% ( Tortoli et al, 2012 ; Sauzullo et al, 2016 ; Lee et al, 2017 ; Bahr et al, 2018 ; Dorman et al, 2018 ; Sarfaraz et al, 2018 ; Sulis et al, 2018 ; Aydemir et al, 2019 ; Kohli et al, 2021 ) and can reach more than 90% only in some body districts ( Mazzola et al, 2016 ). The few data available regarding the Xpert assay performances in FFPE samples are based on a few clinical biopsies and reported a wide range of sensitivities, which were not even concordant (from 97.6% in the case of the Ultra version to 53.2% in the case of the first MTB/RIF assay) ( Seo et al, 2014 ; Du et al, 2019 ; Njau et al, 2019 ; Budvytiene and Banaei, 2020 ; Huang et al, 2020 ). Some of these reports also suggest different sensitivities against the sites of biopsies (i.e., lymph node vs. non-lymph nodes sites) ( Polepole et al, 2017 ).…”

Bronchial brushing Xpert improves the diagnostic efficiency of sputum Xpert in patients with pulmonary tuberculosis

“…Many studies have discovered that Xpert assay is a promising method for the diagnosis of PTB and extrapulmonary TB using several specimens, such as sputum, body fluids, tissues, and others. [11][12][13][14] However, very limited data exist on the Xpert assay using bronchial brushing for the diagnosis of PTB. Our findings show that the Xpert assay has a high diagnostic yield in the combination of sputum and bronchial brushing, and 69.2% of TB cases were identified by Xpert using both sputum and bronchial brushing samples.…”