Identification and Characterization of Phenotypically Occult Multidrug Resistant Mycobacterium Tuberculosis Strains in Pakistan

Abstract

Tuberculosis (TB) is one of the known ancient infectious disease and is still part of the world’s disease burden despite vaccination and the availability of anti-tuberculosis drugs for treatment. As per World Health Organization 2020 report on TB, there are an estimated 10 million TB cases and 1.4 million per annum deaths due to TB disease. This situation of TB is further aggravated due to the development of multidrug resistance (MDR) and extensively drug resistance (XDR) TB. Rifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid for its treatment. The resistance to RIF is used as a surrogate marker for MDR TB diagnosis in new as well as previously treated patients. Pakistan is among the top five high TB burdened countries, even for high MDR TB cases. For the diagnosis of drug resistance (DR) TB, WHO endorsed phenotypic and genotypic assays are used, however, all these methods have discrepancies in the diagnosis of drug resistance. Thus, discordance in results for drug resistance is encountered frequently, which is the leading cause of misdiagnosis and treatment failure outcomes. The current study was designed to identify the extent of discordance in diagnosis based on the results of molecular GeneXpert MTB/ RIF assay and phenotypic rifampicin drug susceptibility testing (DST) on Lowenstein Jensen (LJ) and commercial Bactec MGIT 960 (MGIT). A total of 1986 culture-positive samples out of 6,148 samples were selected for the study which were received from different Programmatic Management of Drug Resistant TB sites, public sector hospitals of Punjab including Islamabad capital territory and Kashmir. The majority of the TB study population was of the age group 15 to 44 years which accounted for 72.2% of the total TB cases. Among all TB patients included in this study, 1876 (94%) were diagnosed with pulmonary TB, whereas 110 (6%) were extrapulmonary TB cases. Out of 1986 TB cases, concordance in the drug susceptibility results of GeneXpert, MGIT, and LJ DST was found in 1760 Mtb isolates. However, discordance was identified in 226(11.4%) isolates in either of the assays when all three assays’ results were compared. xvi In these identified occult and concordant strains on basis of GeneXpert, MGIT, and LJ DST results were Sanger sequenced for the rpoB gene to determine the molecular basis for RIF resistance and discordance results. Sequencing of rpoB gene was performed on selected 516 isolates comprising of 140 strains with discordant and 376 isolates with concordant results. The sequencing of these strains found 49 different insertions, deletions, and single double and triple nucleotide polymorphisms in Rifampicin Resistance Determining Region (RRDR) as well as outside RRDR. Among these 49 identified mutations, 03 were novel mutations in the rpoB gene in these local Mtb strains. The most frequent mutations were detected at codon 450, 445, and 435 of the rpoB gene, which constituted 58%, 14% and 11% prevalence respectively. A high number of mutations (362) were detected in the 81-bp core region of the rpoB gene, whereas 2 different mutations were detected outside RRDR at position 170 in one isolate and codon 491 in five isolates. GeneXpert MTB/RIF assay for RIF resistance results was compared with sequencing, a 10.9 % discordance was observed in these two molecular methods’ results of 516 selected isolates. The 3.2% discordance was present in GeneXpert sensitive isolates which were identified to be resistant after rpoB gene sequencing, while 7.7% discordance results were observed in GeneXpert RIF resistant isolates which were wild type upon Sanger sequencing. The phenotypic assay MGIT DST results upon comparison with sequencing had 24% discordance, where 23.7% of discordant strains were harboring rpoB gene mutations but were RIF susceptible on MGIT DST. The solid-based LJ DST results comparison with sequencing found a discordance of 10.9% which is lower than MGIT DST. Among these 10.6% isolates, which were resistant due to rpoB gene mutations after sequencing but were initially detected as susceptible on LJ DST. Most prevalent nonsynonymous mutations altered a codon which resulted in the change of single amino acid in various isolates and was associated with discordant results, such identified mutations were Leu433Pro in 19/20 isolates and 14/20isolates, Asp435Tyr in 17/21 and 5/21, Leu452Pro in 17//20 and 5/20, His445Asn in 15/18 and 11/18, His445Leu in 8/9 and 0/9 and His445Cys in 2/6 and 1/6 on resistant on MGIT and LJ DST respectively. Leu430Arg change was detected in two isolates, while Ser431Gly, xvii Asn438Lys, and Thr444Ala mutations were found in one isolate each. Also, in the current study mutations were characterized as discordant which were not reported as discordant in previous studies. Whole Genome sequencing was performed on 25 selected isolates including drug susceptible (DS) TB, MDR TB, Pre-XDR, and XRD TB isolates. A large number of mutations were identified in katG, inh A, gyrA, gyrB, embB, pncA, and rss genes, which are responsible for drug resistance development to other anti TB drugs used for the treatment of drug-susceptible and DR TB. Upon comparison of phenotypic drug susceptibility testing results for other than RIF anti TB drugs with WGS, phenotypic and genotypic resistance results were in line but in some cases, discordance in results was recorded. Even for newly introduced anti TB drugs Bedaquiline and clofazimine resistance-conferring mutations in the Rv0678 gene were detected through WGS, which highlights serious concern as intrinsic resistance to these newly approved drugs is present already in the local Mtb strain. To further dissect the impact of mutations in the rpoB gene on this gene encoded DNA dependent RNA polymerase structure, in silico bioinformatics protein prediction tool DynaMut was used. The predicated RNA polymerase structure in most of the identified mutations destabilized protein and increased flexibility of polymerase even binding affinity to the target drug was altered. Phylogenetic analysis for the lineage of Mtb isolates revealed that the majority belonged to Delhi⁄CAS lineage followed by Euro-American Super linage. The discordance between results of different assays was not related to patients’ age, gender, and treatment category or lineage identified. Based on current study findings, among phenotypic methods, LJ DST was found to be less prone to miss rifampicin resistance compared to MGIT DST. Routine WHO endorsed genotypic assay (GeneXpert) used for screening of rifampicin resistance also missed resistance if a mutation in strain was outside RRDR region, hence posing a major setback where strains will be misdiagnosed leading to treatment failures and proliferation of DR strains if were treated as RIF sensitive TB. All three methods (LJ, MGIT, and GeneXpert) had limitations in the drug resistance detection due inability to pick both xviii previously reported as well as newly identified mutations, which in turn will give high discordance results. Phenotypic methods are the existing gold standard for DST of DR TB but were found mostly failed to compete with sequencing technologies for the diagnosis of rifampicin-resistant TB and MDR TB. Based on this study, it can be proposed that sequencing should be endorsed as the gold standard for the diagnosis of DR TB especially suspected cases of MDR TB and XDR TB. It can be recommended after extensive comparison of WHO endorsed various DST assays, globally as well as in Pakistan gold standard method for detection of RIF resistance and MDR should be revisited, and a new evidence-based DST for TB should be adopted for better treatment, management, and reduction of all types of TB