Tuberculosis (TB) continues to be a global health threat and a leading cause of adult mortality by a single infectious disease over the years and is currently is second only to COVID-19 (WHO, 2020a; Woolf et al., 2021). The WHO recommends the directly observed treatment short-course (DOTS) regimen, which involves the use of multiple antibiotics in an intensive phase therapy (Isoniazid; INH, Rifampicin; RIF, pyrazinamide; PZA, and ethambutol; EMB) for 2 months followed by 4 months continuous-phase administration of INH and RIF for the treatment of uncomplicated or drug-sensitive TB (DS-TB) (World Health Organization [WHO], 2017). The intensive phase is aimed at killing the tubercle bacilli as fast as possible to reduce the bacterial load to stop community transmission whereas the continuous-phase aims for complete sterilization of the slow-growing bacilli (World Health Organization [WHO], 2010). Even though TB is manageable with anti-TB drugs, the emergence of drug-resistant (DR) strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of TB, threatens to make the disease untreatable.

Tuberculosis drug resistance include INH-mono-resistant (INH^r) for resistance to only INH, RIF-mono-resistant (RIF^r) for resistance to only RIF (when susceptibility to INH is known), RIF-resistance (RR) for resistance to RIF diagnosed using GeneXpert (when INH susceptibility is not known) multidrug resistance (MDR) for resistance to both INH and RIF. Recently, the definition for extremely drug resistance (XDR), which was MDR with additional resistance to at least one second line aminoglycoside (AMG) and fluoroquinolone (FQ) was changed after the WHO expert consultation (WHO, 2020b) to ‘TB caused by MTBC strain that fulfils the definition of MDR/RR-TB with additional resistance to any FQ and at least one of bedaquiline (BDQ) and linezolid (LZD).’ Similarly, the definition of pre-XDR which was MDR with additional resistance to either any second line FQ or an injectable AMG was also changed to “TB caused by MTBC strain that fulfils the definition of MDR/RR-TB is also resistant to any FQ” after the expert consultation meeting (WHO, 2020b). Therefore, polyresistance (PR) TB can then be defined as INH^r with additional resistance to at least one second line drug. Treatment of DR-TB requires prolonged administration of relatively more toxic, less efficacious medication (WHO, 2012, 2021) extending the time for sputum conversion. This fuels the spread of resistant strains within the community. It was estimated that 71% of bacteriologically confirmed TB cases (5.8 million) in 2020 were tested for RIF resistance, among which 132,222 (2.3%) were MDR/RIF-TB and 25,681 (0.4%) resistant to at least one of AMGs and FQs (WHO, 2021). The threat of MDR-TB is further exacerbated by the emergence XDR-TB which does not respond to the commonly available TB drugs and requires the use of new formulations and specialized regimen including some of the newest TB drugs such as bedaquiline (BDQ) and linezolid (LZD) depending on the specific resistant profiles.

Previous research studies conducted in Ghana identified several MDR-TB and pre-XDR-TB cases (Homolka et al., 2010; Asante-poku et al., 2015; Otchere et al., 2016; Osei-Wusu et al., 2018a). To the best of our knowledge, these previous studies were not linked to program activities to benefit respective patients. This program-linked activity was initiated to screen difficult-to-treat (DTT) TB patients (sputum-smear non-convertors, failed, relapsed, and known DR-TB patients as well as TB patients with history of TB treatment) through a collaboration between the National Tuberculosis Control Program (NTP) and the Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana.

Resistance to anti-tuberculosis drugs is a major threat to achieving the End TB goals (WHO, 2012; Getahun et al., 2013; El Hamdouni et al., 2019). Early diagnosis of TB and ascertaining the drug susceptibility profile of the infecting pathogen are required to initiate an appropriate treatment regimen. However, most TB management protocols around the globe are based on passive case detection leading to late diagnosis with its associated inimical public health implications. We passively screened TB patients in Ghana belonging to at least one of the groups including retreatment, failed treatment (sputum non-converting), defaulted treatment, and known DR-TB.

We found that 58% of cases were resistant to at least one first-line drug (ANY^r) including 16% INH^r, 16.8% RIF^r, and 25.2% MDR. These proportions are significantly higher than what was detected by previous independent studies in Ghana (Otchere et al., 2016; Addo et al., 2017). However, our findings compares with findings from a recent study in Ghana (Sylverken et al., 2021) and others parts of the world (He et al., 2008; Oxlade et al., 2010; World Health Organisation and Regional Office, 2014; Luo et al., 2017; Song et al., 2020; Mbuh et al., 2021) which reported higher MDR rates among retreatment cases compared to new TB cases. New TB cases among the DTT-TB samples were more likely to be susceptible to INH and RIF (p < 0.001) compared to retreatment cases suggesting that higher bacteria load may be the cause of sputum non-conversion after the intensive phase of DOTS but not drug resistance. Such cases normally require extension of the intensive phase of the treatment regimen by at least on month for better clearance of the bacteria (World Health Organization [WHO], 2010).

Our study confirmed katG MT as the main cause of INH-resistance in Ghana, and these samples were more likely to have absence of WT bands with corresponding presence of MT bands compared to rpoB mutant RIF-resistant samples which mostly had presence of MT bands without missing WT bands. This observation suggests that most of the affected TB patients had INH-resistance already fixed in the infecting bacteria while RR had nascently emerged among a sub-population of the bacteria. This finding support the general idea that INH-resistance is the precursor for emergence of MDR-TB (Stagg et al., 2017; O’Donnell, 2018; Srinivasan et al., 2020). Unfortunately, the most recommended rapid TB diagnostic tool (GeneXpert) only detects RR as proxy for MDR-TB. There is therefore the need to consider incorporating detection of INH-resistance into the GeneXpert and/or any other new diagnostic tool to aid in early detection of INH-resistance to curb emergence of MDR-TB.

The MTBDRsl assay targets MT within the “quinolone resistance-determining regions” spanning the gyrA and gyrB loci thus increasing its sensitivity (Sun et al., 2008). We found from our study that all FQ-resistant samples had either gyrA WT band missing or gyrA mutation band present or both with no involvement of the gyrB locus. This observation agrees with the findings of two independent studies (Von Groll et al., 2009; Kabir et al., 2020) which found over 98% of FQ-resistance was caused by gyrA MT alone. The gyrA and gyrB are the two sub-units of the gyrase enzyme which is the target of FQ drugs (Xu et al., 1996). Therefore, the observation of only gyrA MT among FQ-resistant samples is mind bugling. Could it be that the gyrA sub-unit of the bacterial DNA gyrase is potentially the main target of FQ drug or do gyrA MT offer competitive advantage to the bacteria?

There were 2 out of the identified 19 pre-XDR samples which would have been classified as XDR-TB samples by the old WHO definition (MDR with further resistance to FQs and AMGs). These samples had genetic alterations at the gyrA and eis loci (for low-level KAN resistance) compared to the first XDR-TB case identified in Ghana (by the old WHO definition) which had MT at the rrs loci (for resistance to KAN/AMK/CAP/VIO) and gyrA (Osei-Wusu et al., 2018b). These two scenarios clearly have different strains of MTBC which acquired resistance through different evolutionary paths and would require different combination of drugs for treatment (Kambli et al., 2016). Furthermore, the new WHO definitions of XDR and pre-XDR TB (WHO, 2020b) highlight the importance of FQs but not AMGs in the management of MDR/RR TB and the need to prevent emergence and potential spread of FQ resistance which is the precursor for pre-XDR and XDR TB among MDR/RR TB patients. Nevertheless, all the identified AMG-polyresistant samples were associated with the rrs locus without the involvement of the eis locus, suggesting high level resistance to AMGs (Kambli et al., 2016) which cannot be ignored. Nevertheless, the identification of two pre-XDR TB cases at primary diagnosis (indication of potential ongoing transmission of pre-XDR TB) from two different regions of the country is very alarming.

We report the detection of several pre-XDR TB cases in Ghana through passive screening of DTT TB patients. Our study is limited by the fact that we did not have any rapid diagnostic tool for detection of BDQ and/or LZD resistance to determine whether some of the identified 19 pre-XDR samples are XDR-TB samples by the new WHO definition. Nevertheless, the high proportion of second line resistance among retreatment cases call for intensified surveillance and active monitoring of TB patients, especially those at risk of drug resistance. Additionally, the likelihood of an ongoing transmission of pre-XDR TB in Ghana calls for active surveillance for DR-TB. There is also the need for inclusion of BDQ and LZD in existing and new rapid diagnostics to enhance early detection to support an appropriate treatment regimen to help curb the spread of DR-TB. Finally, there is the need for intensified public education on the need to comply with TB treatment regimen for effective treatment toward reduction of DR-TB.

The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The protocols and procedures for this study were reviewed and approved by the institutional review board of the NMIMR, with federal-wide assurance number FWA00001824. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

IO conceptualized the idea, developed methods, provided resources, carried out the research, wrote first draft, and reviewed the manuscript. PM, PA, SO-W, SA, AM, and TA carried out the research and reviewed the manuscript. SY, ED, GT-O, CL, YP, and FB provided resources and reviewed the manuscript. AA-P provided resources, carried out the research, and reviewed the manuscript. SG provided resources, supervised the work, and reviewed the manuscript. DY-M conceptualized the idea, developed methods, provided resources, supervised the work, and reviewed the manuscript. All authors contributed to the article and approved the submitted version.