Xpert mtb rif assay

A single sputum sample per patient was used for the diagnosis of TB using Xpert-MTB/RIF assay (Cepheid, Sunnyvale, CA, USA). Briefly, after sputum was collected, it was mixed with sample reagent buffer in 1:2 (sample: sample reagent buffer) volume ratio. Then, closing it tightly, vortexed for 15 seconds and allowed to stand at room temperature for 10 min. It was again vortexed after 10 min and allowed to stand for 5 min, using the Pasteur pipette provided with the kit >2mL of the (just above 2mL mark on pipette) processed sample was put into the Xpert -MTB/RIF cartridge. Then the cartridge with the specimen was loaded to the Xpert machine. Finally, results were collected from the Xpert computer after 2h [16 ].

CSF specimens were stained and cultured by standard methods for pyogenic bacteria, mycobacteria, and fungi. In the last study [3 (link)], CSF was also tested with the Xpert MTB/RIF assay (Cepheid). Isolates of M. tuberculosis were tested for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin with the mycobacterial growth indicator tube method [15 (link)]. Baseline peripheral blood CD4 cell counts were measured for all HIV-infected adults, using flow cytometry.

Various amounts (0.3 g to 1.0 g) of two stool samples with a soft consistency were used to test different stool processing protocols before testing with the Xpert MTB/RIF assay (Cepheid) for the detection of M. tuberculosis and resistance to rifampin. This testing was done on a GeneXpert instrument (Cepheid), following the instructions of the manufacturer. The protocol for the proposed SOS stool method consisted of the standard protocol for sputum processing for Xpert testing (16 (link), 17 (link)) with a few minor modifications (see details below), i.e., a single release/sedimentation step. In addition, various other stool processing protocols were tested, with the following additional steps: (i) addition of glass beads to improve sample homogenization and potentially increase the release of M. tuberculosis, following the method described by Banada et al. (12 (link)); (ii) dilution with PBS, resembling the TS method described by Andriyoko et al. (15 (link)); and (iii) filtration following the method described by Banada et al. (12 (link)). Also, protocols that consisted of various combinations of these additional steps were evaluated. Figure S1 in the supplemental material shows a schematic overview of the series of laboratory experiments and more details on how these were performed.

Two sputum samples were collected from each patient and examined by Ziehl-Neelsen staining at the local diagnostic units. Smear-positive samples were referred to the NRL at the Chakib Saad Hospital in the capital of Djibouti for testing using the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA). In cases where rifampicin resistance was detected, MTB strains were isolated by liquid culture using the BACTEC^TMMGIT^TM 960 System (MGIT) (BD Bioscience, Erebodegem, Belgium). Positive MTB cultures underwent first-line drug susceptibility testing (DST) in liquid media using the following critical concentrations: rifampicin 1.0 mg/L, isoniazid 0.1 mg/L, ethambutol 5.0 mg/L and streptomycin 1.0 mg/L. All rifampicin-resistant strains and all available rifampicin-susceptible sputum specimens were sent to the TB supranational reference laboratory (SRL) in Milan, Italy for genotypic DST based on next generation sequencing (Supplementary Figure 3). Handling of sputum samples and MTB culture isolates was conducted following essential biosafety measures as recommended by WHO²⁹ . MTB isolates were shipped to SRL Milan following the IATA regulations for UN2814 Infectious Substances affecting humans (Class 6.2).

All patients had a diagnostic lumbar puncture performed to obtain cerebrospinal fluid (CSF) for microbiological and molecular testing at the time of admission to the NCTLD. Microbiologic testing of CSF included Ziehl-Neelsen microscopy and mycobacterial culture on both Löwenstein-Jensen (LJ) solid and Mycobacterial Growth Indicator Tube (MGIT) liquid media. In cases with a positive culture for Mycobacterium tuberculosis, isolates underwent testing with the MTBDRplus line probe assay (Hain Lifescience, Nehren, Germany) and phenotypic DST as previously described [16 (link)]. Starting in April 2015, testing with the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) was implemented for all baseline CSF samples. Approximately 1–4 ml of non-centrifuged CSF was used and the test was performed according to standard procedures. General testing including CSF bacterial cultures, complete blood count and chemistry evaluations were carried out per Georgia NTP guidelines.

The study population consisted of all the participants in the ATI-TB project that were tested for PTB (from here on, “presumptive PTB cases”) by Xpert MTB/RIF assay (Cepheid). Participants included both people tested during the awareness and ACF campaigns (i.e., active-case finding) and the so-called walk-in patients. The latter were people who spontaneously accessed a local health facility because of symptoms (passive-case finding) and got their sputum tested using one of the two GeneXpert machines purchased by the ATI-TB project and installed in Ngong (Kajiado North) and Oltepesi (Kajiado West). These machines were used to test both the locally collected sputum samples and those sent from other health centers of the area. The third GeneXpert machine bought for the project was used as a mobile Xpert MTB/RIF for the ACF activities.