Discordance in Phenotypic and Genotypic Susceptibility Testing for Streptomycin Due to Nonsynonymous/Nonsense/Deletion Frame-Shift Mutations in Gidb Among Clinical Mycobacterium tuberculosis Isolates in Kuwait.

OBJECTIVE: Increasing reports of resistance to newer anti-tuberculosis drugs have prompted the search for other alternative drugs. Streptomycin could be used for the treatment of drug-resistant tuberculosis if susceptibility of Mycobacterium tuberculosis isolate to streptomycin could be accurately detected. We performed phenotypic and genotypic drug susceptibility testing (DST) of 118 M. tuberculosis isolates for streptomycin. MATERIALS AND METHODS: Fifty pansusceptible and 68 multidrug-resistant M. tuberculosis (MDR-TB) isolates were used. Phenotypic DST for streptomycin, rifampicin, isoniazid and ethambutol was performed by mycobacteria growth indicator tube (MGIT) 960 System. Genotypic DST was done by GenoTypeMTBDRplus assay for rifampicin and isoniazid and by PCR-sequencing of rpsL, rrs and gidB genes for streptomycin. MDR-TB isolates were genotyped by spoligotyping. RESULTS: Phenotypic DST identified 50 isolates susceptible to all four drugs (pansusceptible). Sixty-one of 68 MDR-TB isolates were resistant to streptomycin. Genotypic testing for rifampicin and isoniazid yielded expected results. Fifty pansusceptible and 7 streptomycin-susceptible MDR-TB isolates contained no mutation in rpsL or rrs, while 47, 2 and 1 STR-resistant isolate contained rpsL, rrs and rpsL + rrs mutations, respectively. Of the remaining 11 STR-resistant MDR-TB, 9 isolates contained deletion frame-shift/nonsynonymous mutations in gidB. Surprisingly, 13 pansusceptible isolates also contained deletion frame-shift/nonsense/nonsynonymous mutations in gidB. Also, 30 of 68 MDR-TB but only 2 of 50 pansusceptible isolates belonged to the Beijing genotype. CONCLUSIONS: Our data show that, like ifampicin, ethambutol and pyrazinamide, streptomycin also exhibits discordant phenotypic and genotypic DST results for some M. tuberculosis isolates. Hence, streptomycin should be included in therapy regimens only if both phenotypic and genotypic resistance testing indicate susceptibility to avoid amplification of resistance and drug toxicity.

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Kuwait and evaluation of REBA MTB-MDR assay for rapid detection of MDR-TB.

Early detection of Mycobacterium tuberculosis (Mtb) in clinical specimens, its susceptibility to anti-TB drugs and disruption of infection transmission to new hosts are essential components for global tuberculosis (TB) control efforts. This study investigated major Mtb genotypes circulating in Kuwait and evaluated the performance of REBA MTB-MDR (REBA) test in comparison to GenoType MTBDRplus (gMTBDR+) assay for rapid detection of resistance of Mtb to isoniazid and rifampicin (MDR-TB). M. tuberculosis isolates (n = 256) originating predominantly from expatriate patients during a 6-month period were tested by spoligotyping and a dendrogram was created by UPGMA using MIRU-VNTRplus software. Phenotypic drug susceptibility testing (DST) was performed by MGIT 960 system. Genotypic DST for isoniazid and rifampicin was done by REBA and gMTBDR+ assays. Spoligotyping assigned 188 (73.4%) isolates to specific spoligotype international type (SIT) while 68 isolates exhibited orphan patterns. All major M. tuberculosis lineages were detected and EAI, CAS and Beijing families were predominant. Phylogenetic tree showed 131 patterns with 105 isolates exhibiting a unique pattern while 151 isolates clustered in 26 patterns. Fifteen isolates were resistant to one/more drugs. REBA and gMTBDR+ detected isoniazid resistance in 11/12 and 10/12 and rifampicin resistance in 4/5 and 4/5 resistant isolates, respectively. The diversity of SIT patterns are highly suggestive of infection of most expatriate patients with unique Mtb strains, likely acquired in their native countries before their arrival in Kuwait. Both, REBA and gMTBDR+ assays performed similarly for detection of resistance of Mtb to isoniazid and rifampicin for rapid detection of MDR-TB.

Increasing prevalence of resistance to second-line drugs among multidrug-resistant Mycobacterium tuberculosis isolates in Kuwait.

Molecular methods detect genetic mutations associated with drug resistance. This study detected resistance-conferring mutations in gyrA/gyrB for fluoroquinolones and rrs/eis genes for second-line injectable drugs (SLIDs) among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates in Kuwait. Fifty pansusceptible M. tuberculosis and 102 MDR-TB strains were tested. Phenotypic susceptibility testing was performed by MGIT 960 system using SIRE drug kit. GenoType MTBDRsl version 1 (gMTBDRslv1) and GenoType MTBDRsl version 2 (gMTBDRslv2) tests were used for mutation detection. Results were validated by PCR-sequencing of respective genes. Fingerprinting was performed by spoligotyping. No mutations were detected in pansusceptible isolates. gMTBDRslv1 detected gyrA mutations in 12 and rrs mutations in 8 MDR-TB isolates. gMTBDRsl2 additionally detected gyrB mutations in 2 and eis mutation in 1 isolate. Mutations in both gyrA/gyrB and rrs/eis were not detected. gMTBDRslv1 also detected ethambutol resistance-conferring embB mutations in 59 isolates. Although XDR-TB was not detected, frequency of resistance-conferring mutations for fluoroquinolones or SLIDs was significantly higher among isolates collected during 2013-2019 versus 2006-2012. Application of both tests is warranted for proper management of MDR-TB patients in Kuwait as gMTBDRslv2 detected resistance to fluoroquinolones and/or SLIDs in 3 additional isolates while gMTBDRslv1 additionally detected resistance to ethambutol in 58% of MDR-TB isolates.

Correction to: 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.

The original publication of this article [1] contained few erroneous paragraphs and errors in Table 1 and Table 2. The first four paragraphs are in the 'Results' section while the last four paragraphs are in the 'Discussion' section. The errors in Table 1 involve the number of isolates tested for pyrazinamide and pyrazinamide susceptible isolates, ethambutol-susceptible isolates with a mutation and number of resistant isolates with a mutation for streptomycin. The error in Table 2 involves wrong codon number for a mutation in isolate KM17-01 in Cluster XII for gidB gene. The updated informations have been indicated in bold and also refer corrected Tables 1 and 2.

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.

BACKGROUND: Increasing incidence of multidrug-resistant Mycobacterium tuberculosis infections is hampering global tuberculosis control efforts. Kuwait is a low-tuberculosis-incidence country, and ~ 1% of M. tuberculosis strains are resistant to rifampicin and isoniazid (MDR-TB). This study detected mutations in seven genes predicting resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin in MDR-TB strains. Sequence data were combined with spoligotypes for detecting local transmission of MDR-TB in Kuwait. METHODS: Ninety-three MDR-TB strains isolated from 12 Kuwaiti and 81 expatriate patients and 50 pansusceptible strains were used. Phenotypic drug susceptibility was determined by MGIT 460 TB/960 system. Mutations conferring resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin were detected by genotype MTBDRplus assay and/or PCR sequencing of three rpoB regions, katG codon 315 (katG315) + inhA regulatory region, pncA, three embB regions and rpsL + rrs-500-900 regions. Spoligotyping kit was used, spoligotypes were identified by SITVIT2, and phylogenetic tree was constructed by using MIRU-VNTRplus software. Phylogenetic tree was also constructed from concatenated sequences by MEGA7 software. Additional PCR sequencing of gidB and rpsA was performed for cluster isolates. RESULTS: Pansusceptible isolates contained wild-type sequences. Mutations in rpoB and katG and/or inhA were detected in 93/93 and 92/93 MDR-TB strains, respectively. Mutations were also detected for pyrazinamide resistance, ethambutol resistance and streptomycin resistance in MDR-TB isolates in pncA, embB and rpsL + rrs, respectively. Spoligotyping identified 35 patterns with 18 isolates exhibiting unique patterns while 75 isolates grouped in 17 patterns. Beijing genotype was most common (32/93), and 11 isolates showed nine orphan patterns. Phylogenetic analysis of concatenated sequences showed unique patterns for 51 isolates while 42 isolates grouped in 16 clusters. Interestingly, 22 isolates in eight clusters by both methods were isolated from TB patients typically within a span of 2 years. Five of eight clusters were confirmed by additional gidB and rpsA sequence data. CONCLUSIONS: Our study provides the first insight into molecular epidemiology of MDR-TB in Kuwait and identified several potential 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 can identify possible cases of local transmission of MDR-TB in low MDR-TB settings.

Occurrence of disputed rpoB mutations among Mycobacterium tuberculosis isolates phenotypically susceptible to rifampicin in a country with a low incidence of multidrug-resistant tuberculosis.

BACKGROUND: Accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis in clinical specimens and culture isolates to first-line drugs is crucial for diagnosis and management of multidrug-resistant tuberculosis (MDR-TB). Resistance of M. tuberculosis to rifampicin is mainly due to mutations in hot-spot region of rpoB gene (HSR-rpoB). The prevalence of disputed (generally missed by rapid phenotypic DST methods) rpoB mutations, which mainly include L511P, D516Y, H526N, H526L, H526S, and L533P in HSR-rpoB and I572F in cluster II region of rpoB gene, is largely unknown. This study determined the occurrence of all disputed mutations in HSR-rpoB and at rpoB codon 572 in M. tuberculosis strains phenotypically susceptible to rifampicin in Kuwait. METHODS: A total of 242 M. tuberculosis isolates phenotypically susceptible to rifampicin were used. The DST against first-line drugs was performed by Mycobacteria growth indicator tube (MGIT) 960 system. Mutations in HSR-rpoB (and katG codon 315 and inhA-regulatory region for isoniazid resistance) were detected by GenoType MDBDRplus assay. The I572F mutation in cluster II region of rpoB was detected by developing a multiplex allele-specific (MAS)-PCR assay. Results were confirmed by PCR-sequencing of respective loci. Molecular detection of resistance for ethambutol and pyrazinamide and fingerprinting by spoligotyping were also performed for isolates with an rpoB mutation. RESULTS: Among 242 rifampicin-susceptible isolates, 0 of 130 pansusceptible/monodrug-resistant isolates but 4 of 112 polydrug-resistant isolates contained a disputed rpoB mutation. All 4 isolates were also resistant to isoniazid and molecular screening identified additional resistance to pyrazinamide and ethambutol in one isolate each. In final analysis, 2 of 4 isolates were resistant to all 4 first-line drugs. Spoligotyping showed that the isolates belonged to different M. tuberculosis lineages. CONCLUSIONS: Four of 242 (1.7%) rifampicin-susceptible M. tuberculosis isolates contained a disputed rpoB mutation including 2 isolates resistant to all four first-line drugs. The occurrence of a disputed rpoB mutation in polydrug-resistant M. tuberculosis isolates resistant at least to isoniazid (MDR-TB) suggests that polydrug-resistant strains should be checked for genotypic rifampicin resistance for optimal patient management since the failure/relapse rates are nearly same in isolates with a canonical or disputed rpoB mutation.

Prevalence of tuberculosis and multidrug resistant tuberculosis in the Middle East Region.

INTRODUCTION: Prevalence of TB and MDR-TB varies considerably among various regions of World Health Organization (WHO) and also among individual countries within each region. Many Middle Eastern countries have war/civil war-like situations, refugees from war-torn countries or dynamic expatriate population from TB endemic countries which will likely affect the END-TB strategy launched by the WHO in 2015. Areas covered: The data for each of 17 countries comprising the Middle East were analyzed for estimated incidence of TB, number of notified TB cases, mortality rate, and rate of MDR-TB in new and previously treated TB cases as reported by WHO. Data from national surveys or surveillance studies from individual countries were also analyzed for incidence of MDR-TB in new and previously treated TB patients and compared with the estimated data by WHO. Expert commentary: Several Middle Eastern countries have low/intermediate incidence rate and are striving for TB elimination. Reaching pre-elimination (< 1 TB case per 100 000 population) stage will require testing and treatment of latent TB infection in groups at high risk of reactivation and effective treatment of drug-susceptible and drug-resistant TB cases. Large numbers of refugees, expatriate workers, or confounding noncommunicable diseases in some countries pose major challenges in achieving progress toward TB elimination.

Molecular Screening Versus Phenotypic Susceptibility Testing of Multidrug-Resistant Mycobacterium tuberculosis Isolates for Streptomycin and Ethambutol.

Proper management of multidrug-resistant tuberculosis (MDR-TB) requires accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis isolates to other (ethambutol [EMB], pyrazinamide, and streptomycin [SM]) first-line drugs. This study compared the performance of Mycobacterium Growth Indicator Tube (MGIT) 960 system for DST of MDR-TB isolates with polymerase chain reaction (PCR) sequencing of embB, rpsL, and rrs genes for detecting resistance to EMB and SM. MDR-TB strains (n = 60) and 25 pansusceptible M. tuberculosis isolates collected during 2011-2016 were tested. Phenotypic DST was performed by MGIT 960 system by using SIRE drug kit. EMB and SM resistance-conferring mutations in embB and rpsL+rrs genes, respectively, were detected by PCR sequencing. No mutations were detected in pansusceptible isolates. Among 60 MDR-TB strains, 35 of 40 SM-resistant and none of 20 SM-susceptible isolates contained rpsL and/or rrs mutations (κ = 0.82, very good agreement). However, all 18 EMB-resistant MDR-TB strains and 33 of 42 EMB-susceptible MDR-TB strains contained an embB mutation (κ = 0.14, poor agreement). Thus, 40 of 60 (67%) and 35 of 60 (58%) isolates were resistant to SM (p = 0.451), while 18 of 60 (30%) and 51 of 60 (85%) isolates were resistant to EMB (p = 0.000) by MGIT 960 system and PCR sequencing, respectively. MGIT 960 system showed acceptable performance for DST for SM; however, it performed poorly for EMB as many MDR-TB strains with embB mutations, which confer low-level resistance to EMB, were detected as EMB susceptible. Molecular screening for resistance-conferring mutations in embB gene is thus superior to MGIT 960 system when accurate EMB susceptibility results are needed for proper management of MDR-TB patients.

Variations in the occurrence of specific rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolates from patients of different ethnic groups in Kuwait.

BACKGROUND & OBJECTIVES: Frequency of resistance-conferring mutations vary among isoniazid- and ethambutol-resistant Mycobacterium tuberculosis isolates obtained from patients of various ethnic groups. This study was aimed to determine the occurrence of specific rpoB mutations in rifampicin-resistant M. tuberculosis isolates from tuberculosis patients of various ethnic groups in Kuwait. METHODS: Rifampicin-resistant M. tuberculosis isolates (n=119) from South Asian (n=55), Southeast Asian (n=23), Middle Eastern (n=39) and other (n=2) patients and 107 rifampicin-susceptible isolates were tested. Mutations in rpoB were detected by DNA sequencing. Polymorphisms at katG463 and gyrA95 were detected by PCR-RFLP for genetic group assignment. RESULTS: None of rifampicin-susceptible but 116 of 119 rifampicin-resistant isolates showed rpoB mutation(s). Mutations among isolates from South Asian patients were distributed at rpoB516 (20%), rpoB526 (24%) and rpoB531 (27%) while 78 and 51 per cent of isolates from Southeast Asian and Middle Eastern patients, respectively, contained a mutated rpoB531. All isolates with rpoB N-terminal and cluster II mutations were obtained from Middle Eastern and South Asian patients. Most isolates from South Asian (84%) and Southeast Asian (70%) patients belonged to genetic group I while nearly all remaining isolates belonged to genetic group II. Isolates from Middle Eastern patients were distributed among genetic group I (46%), genetic group II (33%) and genetic group III (21%). INTERPRETATION & CONCLUSIONS: The occurrence of specific rpoB mutations varied considerably in rifampicin-resistant M. tuberculosis isolates obtained from patients of different ethnic groups within the same country. The present data have important implications for designing region-specific rapid methods for detecting majority of rifampicin-resistant strains.

First report of molecular detection of fluoroquinolone resistance-associated gyrA mutations in multidrug-resistant clinical Mycobacterium tuberculosis isolates in Kuwait.

BACKGROUND: Nearly 5% of all Mycobacterium tuberculosis strains worldwide are resistant at least to rifampicin and isoniazid (multidrug-resistant tuberculosis, MDR-TB). Inclusion of a fluoroquinolone and an injectable agent (kanamycin, amikacin or capreomycin) in multidrug therapy is crucial for proper treatment of MDR-TB. The incidence of MDR-TB in Kuwait is ~1%. MDR-TB strains additionally resistant to fluoroquinolones and injectable agents are defined as extensively drug-resistant (XDR-TB) strains and have been detected in >55 countries. Infections with XDR-TB strains have very poor prognosis. This study detected the occurrence of gyrA mutations associated with fluoroquinolone resistance among MDR-TB strains in Kuwait. FINDINGS: Direct DNA sequencing of quinolone resistance-determining region of gyrA gene was performed to detect fluoroquinolone resistance-associated mutations in 85 MDR-TB strains isolated from 55 TB patients and 25 pansusceptible M. tuberculosis strains. For isolates exhibiting gyrA mutations, 3'-end of rrs (16S rRNA) was sequenced for the detection of XDR-TB. Fingerprinting of fluoroquinolone resistant MDR-TB strains was performed by detecting mutations in three (81 bp hot-spot, N-terminal and cluster II) regions of rpoB, katG codon 315 and inhA-regulatory region, polymorphisms at gyrA codon 95 and katG codon 463 by DNA sequencing and by double-repetitive-element PCR for determining strain relatedness. None of the pansusceptible but six of 85 MDR-TB strains contained gyrA mutations. Only gyrA codon 94 was mutated in all six (D94A in one and D94G in five) strains. Three of six mutant strains were recovered from the same patient while three other strains represented individual patient isolates. Fingerprinting studies identified all individual patient isolates as epidemiologically distinct strains. All six strains with a gyrA mutation contained wild-type rrs sequence. CONCLUSIONS: Although fluoroquinolones are generally not used for chemotherapy of TB and drug susceptibility testing for second-line drugs is not carried out in Kuwait, four of 55 (7%) individual patient MDR-TB strains contained mutations in gyrA gene. The data advocate routine drug susceptibility testing for this important second-line drug for proper management of MDR-TB in Kuwait. Lack of mutations in 3'-end of rrs gene that confer resistance to injectable agents reduce the likelihood of occurrence of XDR-TB, at present, in Kuwait.

Comparison of performance of two DNA line probe assays for rapid detection of multidrug-resistant isolates of Mycobacterium tuberculosis.

Infections with multidrug-resistant (resistant at least to rifampicin, RIF and isoniazid, INH) strains of Mycobacterium tuberculosis (MDR-TB) are associated with high case fatality rates. Rapid identification' of MDR-TB strains is important for early institution of appropriate therapy. Two DNA line probe assays, GenoType MTBDR (GT-MTBDR) and INNO-LiPA Rif. TB (INNO-LiPA) were compared for their abilities to detect resistance to INH and RIF in 80 M. tuberculosis isolates. The test results were compared to those obtained by conventional drug susceptibility testing (DST), DNA sequencing and/or PCR-restriction fragment length polymorphism (RFLP) analysis of regions of interest of M. tuberculosis genome. Compared to the DST and katG codon 315 PCR-RFLP results, GT-MTBDR test results were concordant for INH resistance for 63 of 80 (78.7%) isolates. For RIF resistance, GT-MTBDR and INNO-LiPA test results were concordant with DST for 74 of 80 (92.5%) and 76 of 80 (95%) strains, respectively. The GT-MTBDR test results correlated with sequencing results for 77 of 80 (96.2%) while INNO-LiPA results for 79 of 80 (98.7%) isolates. Both the tests are useful for rapid detection of MDR-TB strains, however, GT-MTBDR assay offers the advantage of detecting the resistance to both INH and RIF simultaneously when MDR-TB is suspected.