Comparison of Two Different Real-Time PCR Systems in Postmortem Diagnosis of Tuberculosis in Paraffin-Embedded Tissues

Abstract

Tuberculosis (TB) is one of those infections with high morbidity and mortality in all around the world. Hundreds of people died from this disease without diagnosed or due to resistant strains in Turkey. Therefore, it is important to identify postmortem cases who have died from tuberculosis. Molecular methods have been widely used as well as conventional methods in the diagnosis of tuberculosis. The aim of this study was to compare the two different real-time polymerase chain reaction (Rt-PCR) system in the postmortem diagnosis of Mycobacterium tuberculosis infections in paraffin-embedded tissues. A total of 40 paraffin-embedded tissue samples [lung (n= 35), brain (n= 2), heart (n= 2), lymph node (n= 1)] in which histopathologic findings consistent with TB (necrotizing granulomatous inflammation, gelatinous caseous pneumonia, necrotic fibrous nodul) obtained from 37 autopsy cases (31 male, 6 female; age range: 25-85 yrs) were included in the study. Paraffin-embedded tissues were deparafinized with xylene and ethyl alcohol and then DNA isolation was done with QIAsymphony DSP Virus/Pathogen Midi kit in the QIAsymphony device. DNA amplification process was performed by Rt-PCR using the kit Artus® M. tuberculosis RG-PCR in the Rotor-Gene® Q device (Qiagen, Germany). Likewise, after deparafinization process, samples placed in the cartridge and isolation and Rt-PCR was performed by Xpert® MTB/RIF (Cepheid, USA) system, simultaneosly. Seventeen and 20 out of the 40 paraffin-embedded tissues yielded positive results with Qiagen and Xpert system, respectively. M.tuberculosis DNA was found positive in 13 (32.5%) and negative in 16 (40%) of the samples by both of the systems, exhibiting 72.5% (29/40) of concordance. On the other hand, seven (17.5%) samples that were positive with Xpert system yielded negative result with the Qiagen, while four (10%) samples that were positive with Qiagen yielded negative result with the Xpert system. Of the 20 positive cases detected with Xpert MTB/RIF system, 15 were found rifampicin-susceptible, and three were rifampicin-resistant. In two samples in which M. tuberculosis DNA was low positive, rifampicin resistance could not be detected. The identification of M.tuberculosis infections in postmortem cases will contribute epidemiological data in Turkey. In these cases, effective sampling and diagnosing of M.tuberculosis infections by acid-fast stain and culture methods are crucial. However, in cases without microbiological sampling the detection of M.tuberculosis DNA in paraffin-embedded tissues with PCR, although there are differences between PCR systems has diagnostic value. In conclusion, our data indicated that Xpert MTB/RIF system is more favourable to detect M.tuberculosis DNA in paraffin-embedded tissues, with the advantages of determination of rifampicin resistance, and detection of more positive results within a shorter time.

Magnetic Resonance Imaging and GeneXpert: A Rapid and Accurate Diagnostic Tool for the Management of Tuberculosis of the Spine

“…By combining MRI with GeneXpert, we were able to achieve a considerably high sensitivity of 97.9% for the detection of Mtb in cases of tuberculosis of the spine. Recent literature on GeneXpert supports our findings that the sensitivity for the detection of Mtb and rifampicin resistance using this method can be as high as 95% and 100%, respectively [1011]. However, there are no references in the literature regarding the accuracy of combining MRI and GeneXpert for the diagnosis of tuberculosis of the spine.…”

“…Additionally, GeneXpert is more likely to detect Mtb DNA than traditional PCR, with the added advantage of also determining rifampicin resistance [11]. A delay in diagnosis and the failure to detect drug resistance are major hurdles involved in the treatment of tuberculosis of the spine even today.…”

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

“…3 The sensitivity of Xpert MTB/RIF in our study was comparable to, though slightly higher (53.2% vs 50%) than, a previous study, in which the performance of the QIAGEN artus M. tuberculosis RG PCR assay was compared to Xpert MTB/RIF among 40 FFPE tissues with histopathological features consistent with tuberculosis. 26 Similarly, the sensitivity of Xpert MTB/RIF in our study for lymph node and non-lymph node specimens (53.1% and 53.1%) was higher than that found by Polepole et al (30% and 35%). 20 In further support of the usefulness of Xpert MTB/RIF, its performance was found to be better than ZN in specimens from patients reported to have tuberculosis in other sites and HIV infection.…”

The role of Xpert MTB/RIF in diagnosing pulmonary tuberculosis in post-mortem tissues

“…These findings also support those of a recent autopsy study showing that all TB cases diagnosed through histopathological examination were also Xpert positive in postmortem tissues, although that study did not use a conventional method to confirm TB 20 . However, another study by Yagmur and colleagues comparing real time PCR vs Xpert in paraffin-embedded tissues showed limited sensitivity of both tests (against TB defined by histopathologic findings), which could be attributable to the method of preserving the samples 21 . Although of small sample size, some reports have shown the good performance of Xpert in non-liquid specimens, such as lymphadenopathies 22 , joint and bone 23 , or other tissues 24 25 .…”