Molecular characterization of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates identifies local transmission of infection in Kuwait, a country with a low incidence of TB and MDR-TB

Tuberculosis (TB) is a major infectious disease of global proportions. According to the annual surveys conducted by World Health Organization (WHO), an estimated 10 million new active TB diseases cases and 1.6 million deaths occurred in 2017, making TB as the leading cause of death from a single infectious agent [1]. Near 87% of all TB cases occurred in 30 high-TB-burden countries with nearly 67% of all cases occurring in only eight (India, China, Indonesia, Philippines, Pakistan, Nigeria, Bangladesh and South Africa) countries. In high-TB-burden countries, active TB disease cases usually occur as a result of recent infection or reinfection while in low-TB-incidence countries, most active disease cases occur as a result of reactivation of latent infection acquired few to several years earlier [2‐4]. Most of the TB deaths recorded in recent years have been attributed to drug-resistant (DR) TB [1]. Worldwide, 558,000 people developed TB in 2017 that was resistant to rifampicin, and of these, nearly 457,000 (82%) were inflicted with Mycobacterium tuberculosis strains resistant at least to rifampicin and isoniazid, the two most effective first-line drugs (MDR-TB) [1]. The MDR-TB in resource-limited settings is difficult to treat due to lengthy, more expensive and more toxic treatment regimens which lead to higher rates of clinical failure and disease relapse [5, 6]. MDR-TB is also a risk factor for the development of extensively drug-resistant TB (XDR-TB), infection with MDR-TB strains additionally resistant to a fluoroquinolone and an injectable anti-TB drug, which is even more difficult to treat than MDR-TB [1, 5, 6]. Globally, treatment success rates for fully susceptible TB, MDR-TB and XDR-TB have been estimated as nearly 95%, 55% and 28%, respectively [1]. Rapid and accurate laboratory diagnosis of MDR-TB is crucial for effective treatment, which will also limit further transmission of MDR-TB and evolution of XDR-TB [1, 5].

The evolution of drug resistance in clinical M. tuberculosis strains is mainly due to chromosomal mutations in target genes [5, 7]. The WHO now recommends that drug susceptibility testing (DST) of M. tuberculosis isolates should be carried out for all patients with TB to guide treatment decisions and to improve outcome [1]. Phenotypic DST methods are time-consuming as they require several days to few weeks to report results and are also not widely available [8]. Recent studies have shown that gene sequencing studies reliably predict susceptibility or resistance to first-line anti-TB drugs and are even superior to phenotypic-based DST methods for some first-line drugs [9‐13].

Kuwait, an Arabian Gulf country in the Middle East, is a low TB incidence (~ 24 cases/100 000 population) country [14, 15]. The expatriates account for nearly 75% of the total population of Kuwait. Although all expatriates are screened at the time of entry into Kuwait for the absence of active TB disease, > 80% of all TB cases and > 95% of DR-TB and MDR-TB cases occur among expatriate patients, mainly originating from TB endemic countries of South Asia (Bangladesh, India, Pakistan, etc.), Southeast Asia (mainly Philippines and Indonesia) and Africa [16‐19]. The diversity of mutations and fingerprinting profiles among multidrug-resistant M. tuberculosis isolates have previously shown that most expatriate patients developed active disease in Kuwait due to reactivation of previously acquired infection [17, 18]. Until recently, rifampicin-resistant TB/MDR-TB among Kuwaiti subjects were infrequently detected [16‐19] and transmission of DR-TB was rarely reported in Kuwait [20]. A fourfold increase in the detection of MDR-TB among Kuwaiti subjects was noted in recent years (2014–2017); however, the factors responsible for this sudden increase remained unknown and transmission of MDR-TB within Kuwait was not apparent from routine surveillance studies. This study performed detailed molecular characterization of all MDR-TB strains collected during a 12-year period (2006 to 2017) to detect mutations in seven major gene targets (rpoB, katG, inhA, pncA, embB, rpsL and rrs) conferring resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin. Fingerprinting of all isolates was performed by spoligotyping. The sequence data obtained from multiple loci were also used to generate phylogenetic tree to ascertain strain relatedness (multi-locus sequence analysis, MLSA). Data from spoligotyping and MLSA were combined to define cluster(s) of isolates possibly reflecting local transmission of infection within Kuwait. Sequence data for two additional loci (gidB and rpsA) known to carry phylogenetic polymorphisms [21, 22] were also obtained for cluster isolates obtained within a short time frame (nearly 2 years) to confirm their close genetic relatedness.

The TB control in low incidence settings involves molecular fingerprinting of M. tuberculosis isolates to detect unsuspected outbreaks which also indicates possible epidemiological links between TB patients and differentiates reactivated TB from newly acquired infection [1, 33]. Molecular fingerprinting techniques for M. tuberculosis have evolved from IS6110-based restriction fragment length polymorphism to mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing with/without spoligotyping and more recently, to whole genome sequence (WGS) comparisons [34‐38]. Since whole genome comparisons are not yet standardized, core genome multi-locus sequence typing (cgMLST) has been developed to facilitate high-resolution fingerprinting of clinical M. tuberculosis isolates to trace recent transmission of infections [38, 39]. Genomic sequence-based scanning for drug resistance-associated mutations has also been shown to be nearly comparable to MIRU-VNTR typing [40].

More than 80% of all TB cases and > 95% of DR-TB and MDR-TB cases in Kuwait occur among expatriates [14‐19]. Unexpectedly, several cases of MDR-TB were diagnosed among Kuwaiti subjects during recent (2014–2017) years, and it was not known whether any of these also resulted from cross-transmission of infection or infection from a common source. In this retrospective study, we performed molecular characterization of all available MDR-TB strains collected over a 12-year period to catalog mutations conferring resistance to first-line drugs to determine the feasibility of using commercial molecular methods for rapid diagnosis of MDR-TB in Kuwait. We also employed sequence-based scanning of seven loci to determine relatedness among MDR-TB strains. Further fingerprinting by spoligotyping combined with sequence data for additional loci known to display phylogenetic polymorphisms and other relevant epidemiological information were used to track transmission of MDR-TB among TB patients in Kuwait.

Our data showed that 91 of 93 (97.8%) MDR-TB strains contained a mutation in rpoB hot spot region and S456L mutation was most common, found in 66 of 93 (71%) isolates which is consistent with published reports from different geographical settings [7, 41‐43]. Only three MDR-TB strains contained double rpoB mutations which is also in line with limited previous exposure of TB patients in Kuwait to anti-TB drugs [41, 43]. Similarly, mutations at katG315 and inhA-RR were detected in 80 of 93 (86.0%) and 16 of 93 (17.2%) MDR-TB strains, respectively, which are consistent with the reported worldwide occurrence of these mutations [7, 41‐43]. Altogether, 91 of 93 (98%) MDR-TB strains contained a mutation at katG315 and/or inhA-RR. Of the remaining two isolates, one isolate contained S94A mutation in inhA which is known to confer resistance to isoniazid [44]. Taken together, our data show that rapid molecular diagnostic tests (such as GenoType MTBDRplus assay and targeted PCR sequencing) that interrogate rpoB hot spot region and katG315 + inhA-RR will have a sensitivity of 98% for rifampicin resistance detection, 98% for isoniazid resistance detection and 96% for MDR-TB in Kuwait with 100% specificity. The higher occurrence of S456L mutation in rpoB and S315T mutation in katG in our MDR-TB strains is also consistent with the low fitness cost associated with these specific mutations [45].

Ethambutol, a first-line drug, may be included in treatment regimens for MDR-TB provided the isolate is susceptible to this drug. Streptomycin is no longer considered a first-line drug and is usually not used in treatment regimens due to high rates of resistance in M. tuberculosis isolates globally [5, 6, 46]. Resistance-conferring mutations in rpsL and/or rrs gene was detected in majority (49 of 59, 83%) of streptomycin-resistant but not in any streptomycin-susceptible MDR-TB strain, while mutations in embB gene were detected in both ethambutol-resistant and ethambutol-susceptible MDR-TB strains, as described in our previous studies [23, 28].

Pyrazinamide is used for the treatment of both drug-susceptible TB and MDR-TB [5, 6, 46]. The in vitro susceptibility testing of pyrazinamide is technically challenging and often unreliable due to well-known problems (size of the test inoculum, no growth of some strains in drug-free tube under acidic conditions etc.) recognized in previous studies [47‐49]. Phenotypic DST results for pyrazinamide were available for only 47 of 93 MDR-TB strains, while the remaining 46 isolates failed to grow at lower pH. No pncA mutation was detected in 50 pansusceptible strains. Analysis of 93 MDR-TB strains showed that 30 of 36 MDR-TB strains phenotypically resistant to pyrazinamide and 23 of 46 isolates for which DST data for pyrazinamide was not available contained a mutation in pncA while all 11 MDR-TB strains phenotypically susceptible to pyrazinamide contained wild-type sequence for pncA. The −11 A/G mutation in the regulatory region was most common which is known to reduce the expression of pyrazinamidase [29, 50]. Seven isolates contained insertion/deletion frame shift mutations which will also lead to an inactive pyrazinamidase. Non-synonymous, high/very high confidence resistance mutations with/without additional mutation in regulatory region were observed in 29 isolates which have also been detected in other studies [7, 29, 49]. Our data showing a resistance-conferring mutation in 30 of 36 (83%) of pyrazinamide-resistant and in none of 11 pyrazinamide-susceptible MDR-TB strains support previous observations that pncA mutations may be used as a surrogate marker of pyrazinamide resistance in M. tuberculosis [7, 29, 41, 50, 51].

Spoligotyping data showed that Beijing genotype was most common, shared by 32 of 93 (34%) MDR-TB strains in Kuwait which is expected since this family is known to be strongly associated with multidrug resistance [52]. The occurrence of this genotype was significantly higher in isolates resistant to all four SIRE drugs. The Beijing genotype is also common among drug-resistant/MDR-TB strains from neighboring countries [53, 54]. The occurrence of nine unique (orphan) spoligotyping patterns not reported previously reflects genetic diversity among MDR-TB strains in Kuwait which is not surprising since 81 of 93 (87%) MDR-TB strains were obtained from expatriate patients originating from 11 different countries who were TB-free at the time of their entry into Kuwait, thereby implying that most of these cases likely represent reactivation TB.

The phylogenetic tree generated from concatenated sequence data from partial or complete sequencing of seven (rpoB, katG, inhA, pncA, embB, rpsL and rrs) loci identified 42 isolates in 16 clusters (labeled as Cluster I to Cluster XVI) which could likely represent cross-transmission of infection or infection from a common source. This approach has previously been shown to be nearly comparable to the 24-loci-based MIRU-VNTR typing [40, 55, 56]. The first case in each cluster was considered as the ‘index case’ and an arbitrary window period of nearly 2 years was used to define epidemiologically related strains since reactivation of latent infection usually occurs during the first 2 years (even though this period of latency could also be much longer) after infection with M. tuberculosis [2]. The two isolates in five clusters (Cluster V, VI, IX, XIII and XIV) were genetically unrelated strains by spoligotyping. Similarly, at least some of the isolates in Cluster I and Cluster IV were genetically unrelated strains by spoligotyping while the two isolates in Cluster VII were separated by a much longer time (6 years) than is normally expected. However, isolates in eight clusters (Cluster II, III, VIII, X, XI, XII, XV and XVI) belonged to the same spoligotype and were obtained within a short time span (nearly 2 years) from each other.

Further analysis of gidB and rpsA carried out for selected cluster isolates showed that all isolates in five clusters (Cluster III, VIII, XI, XV and XVI) were genotypically identical strains, highly suggestive of cross-transmission of infection among these patients or infection from a common source. Also, two of three isolates in Cluster X were identical. The two isolates in Cluster XII were also very closely related, with the second isolate (KM17-01) displaying an additional mutation (L95F) in gidB which is considered as a hot spot for mutations in the M. tuberculosis genome [21, 57]. In a recent study, Appelgren et al. [58] investigated transmission of pre-XDR M. tuberculosis infection in a healthcare worker in France by gene-scanning of multiple loci involved in conferring resistance to anti-TB drugs and MIRU-VNTR typing. The authors concluded that the healthcare worker had contacted the infection from a patient that displayed nearly identical mutation and MIRU-VNTR patterns. More elaborate sequence comparisons derived from WGS or cgMLST have also identified transmission of infection in low-TB-incidence settings [59‐61].

Due to the retrospective nature of our study, investigations to confirm the transmission of infection and identification of epidemiological links and contact tracing were delayed and/or were unsuccessful as most of the TB patients, being expatriate subjects, had left the country, after initial treatment objective (sputum smear-negative status) was achieved. The epidemiological investigations for Kuwaiti patients in Cluster XI were also incomplete as the first case did not appear to be the index case as this patient had extra-pulmonary TB and no obvious connection was apparent between the six subjects during contact tracing. It is probable that the index case was an expatriate patient who is not included in this study and had left the country before the cases described here were characterized. Even WGS comparisons sometimes fail to identify transmission of TB in a setting with a high proportion of migrant (expatriate) cases [62].

Our study has a few limitations. (i) Due to retrospective nature of the study, all MDR-TB isolates collected during the study period were not included and epidemiological investigations and contact tracing were delayed. Since expatriate TB patients are sent back to their respective country after initial treatment objective (sputum smear-negative status) was achieved, some patients could not be contacted as they had left the country. (ii) Molecular fingerprinting of the isolates, particularly cluster isolates, was not performed by MIRU-VNTR typing or by more discriminatory WGS or cgMLST.

In conclusion, our data show that rapid molecular diagnostic tests that interrogate hot spot region of rpoB and katG315 + inhA-RR will have a sensitivity of 98% for rifampicin resistance detection, 98% for isoniazid resistance detection and 96% for MDR-TB in Kuwait. Our data also showed that 53 of 93 (57%) MDR-TB strains contain pncA mutations and are thus additionally resistant to pyrazinamide. Our study provides first insight into molecular epidemiology of MDR-TB in Kuwait and identified at least five clusters of local transmission of MDR-TB involving 2–6 subjects which had escaped detection by routine surveillance studies. Prospective detection of resistance-conferring mutations in major gene targets and molecular fingerprinting studies can identify possible cases of local transmission of MDR-TB in low MDR-TB settings which can then be investigated for epidemiological linkage and contact tracing to arrest further transmission of infection.