**1. Background**

In 2017, 10 million people fell ill with tuberculosis (TB) and 1.6 million people died of TB [1]. In the same year, an increase in cases of rifampicin monoresistant and multidrug resistant TB (MDR-TB defined as TB that is resistant to rifampicin and isoniazid) from 490,000 in 2016 to 558,000 in 2017 was observed [1]. The increasing numbers of rifampicin and MDR-TB cases poses a risk to TB control programs throughout the world [2]. Rifampicin monoresistance is considered to be a precursor to MDR-TB and there are often concerns about rifampicin monoresistant TB patients acquiring MDR-TB [3,4]. The standardized World Health Organization (WHO) MDR-TB treatment regimen recommends the use of second-line injectable drugs (SLIDs) in combination with flouroquinolones as part of the standardized MDR-TB treatment regimen [2]. The resistance to a fluoroquinolone and a SLID negatively impacts treatment outcome and has been defined as extensively drug resistant TB (XDR-TB) [5–7]. MDR-TB in combination with resistance to either a fluoroquinolone or a SLID has been termed Pre-XDR-TB.

The resistance to fluoroquinolones is usually caused by point mutations in the quinolone resistance determining region (QRDR) of the gene encoding subunit A or B of the deoxyribonucleic acid (DNA) gyrase gene (*gyrA* or *gyrB*) [8–10]. In the *gyrA* gene, the resistance mutations are commonly found in codons 85 to 96, whereas for the *gyrB* gene, they are found in codons 472 and 510 [5,6]. The mutations between codon 1400 and 1500 in the *rrs* gene are often associated with resistance to SLIDs such as capreomycin, kanamycin and amikacin [5]. The timely detection of resistance to SLIDs remains critical for optimizing treatment to improve the treatment outcome as well as directing infection control measures to halt the transmission of drug resistant TB [7,11]. Diagnostic assays, such as the Hain GenoType MTBDR*sl* line probe assay (Hain Lifescience, Germany), have been endorsed by WHO for the rapid detection of second-line drug resistance [11]. The MTBDR*sl* test is based on the DNA strip technology which has three steps: DNA extraction, multiplex polymerase chain reaction (PCR) amplification and reverse hybridization [12]. This assay has proven to be reliable for rapidly detecting resistance to second-line drugs [13] and has been implemented in 28 countries in Africa [14]. Monitoring drug resistance with the help of such assays and evaluating treatment outcomes may help improve managemen<sup>t</sup> of TB [15,16].

This study sought to determine the level of resistance to second-line drugs among rifampicin monoresistant, isoniazid monoresistant and multi-drug resistant TB cases in Botswana using the Hain genotype MTBDR*sl* Version 2.0 and to assess patient treatment outcomes.
