TB testing strategy and laboratory procedures

The TB testing strategy of the study is summarised in figure 1. The initial microbiological diagnosis of TB was performed using an in-house real-time PCR targeting Mycobacterium tuberculosis (TB-PCR). TB-PCR was performed in all lung samples obtained at CDA, independently of the presence or absence of histological lesions, and in any other organ showing histological lesions suggestive of TB (granulomatous inflammatory reaction and/or caseous necrosis). In addition, in HIV-positive patients, TB was routinely tested by TB-PCR in all central nervous system and cerebrospinal fluid (CSF) samples (independently of the presence or absence of lesions). Histological testing for TB included Ziehl–Neelsen staining when TB was suspected on the haematoxylin and eosin-stained slides.

Algorithm for determination of tuberculosis (TB) diagnosis used in samples from complete diagnostic autopsies. CNS: central nervous system; CSF: cerebrospinal fluid; M. tuberculosis: Mycobacterium tuberculosis.

In addition, the Xpert MTB/RIF Ultra assay (hereafter Xpert Ultra) was performed in 1) any organ with histological lesions suggestive or compatible with TB and positive TB-PCR (cases with TB disease) and 2) in all deaths in which there was microbiological–histological discordance (histological lesions suggestive of TB with negative TB-PCR or positive TB-PCR without histological lesions suggestive of TB).

Tissue samples for Xpert Ultra were thawed and homogenised using a hand-held rotor-stator homogeniser (Qiagen) in ATL lysis buffer (Qiagen). 100 µL of the homogenised tissue sample was added to 300 µL of saline solution. The resulting 400 µL of sample were mixed with 1600 µL of Xpert Ultra Sample Reagent and then loaded into the cartridge. In house TB-PCR was performed using the procedures described by Espasa et al. [23].