Author Correction: Characterization of rifampicin-resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa, Pakistan.

The original version of this Article contained an error in the spelling of the author Otavio Cabral-Marques which was incorrectly given as Otavio Marques Cabral. The original Article has been corrected

Characterisation of drug-resistant Mycobacterium tuberculosis mutations and transmission in Pakistan

Tuberculosis, caused by Mycobacterium tuberculosis, is a high-burden disease in Pakistan, with multi-drug (MDR) and extensive-drug (XDR) resistance, complicating infection control. Whole genome sequencing (WGS) of M. tuberculosis is being used to infer lineages (strain-types), drug resistance mutations, and transmission patterns—all informing infection control and clinical decision making. Here we analyse WGS data on 535 M. tuberculosis isolates sourced across Pakistan between years 2003 and 2020, to understand the circulating strain-types and mutations related to 12 anti-TB drugs, as well as identify transmission clusters. Most isolates belonged to lineage 3 (n = 397; 74.2%) strain-types, and were MDR (n = 328; 61.3%) and (pre-)XDR (n = 113; 21.1%). By inferring close genomic relatedness between isolates (< 10-SNPs difference), there was evidence of M. tuberculosis transmission, with 55 clusters formed consisting of a total of 169 isolates. Three clusters consist of M. tuberculosis that are similar to isolates found outside of Pakistan. A genome-wide association analysis comparing ‘transmitted’ and ‘non-transmitted’ isolate groups, revealed the nusG gene as most significantly associated with a potential transmissible phenotype (P = 5.8 × 10–10). Overall, our study provides important insights into M. tuberculosis genetic diversity and transmission in Pakistan, including providing information on circulating drug resistance mutations for monitoring activities and clinical decision making

Spoligotyping analysis of Mycobacterium tuberculosis in Khyber Pakhtunkhwa area, Pakistan

Sajid Ali,1 Muhammad Tahir Khan,2 Khan Anwar Sheed,3 Muhammad Mumtaz Khan,4 Fariha Hasan11Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan; 2Department of Bioinformatics and Biosciences, Capital University of Science and Technology, Islamabad, Pakistan; 3Provincial TB Reference Laboratory, Provincial TB Control Program, Khyber Pakhtunkhwa, Pakistan; 4Department of Microbiology, University of Haripur, Haripur, PakistanBackground: Spoligotyping is a reproducible, reverse hybridization approach for genotyping of Mycobacterium tuberculosis complex (MTBC). Molecular typing of MTBC is helpful for understanding and controlling tuberculosis epidemics.Methods: Spoligotyping was performed on 166 clinical isolates of Mycobacterium tuberculosis (MTB) collected from 25 districts of Khyber Pakhtunkhwa, Pakistan. Results were compared to SITVIT2, an online database developed by the Institut Pasteur de la Guadeloupe, France.Results: Spoligotyping results showed that 145 strains (88%) displayed known patterns while 21 (12%) were new. Lineage 3/Central Asian strain (L3/CAS) was the predominant family (73%, &chi;2=19.9, P=0.001), followed by L2/Beijing (5.4%) and L4 (4.2%). L3/CAS1-Delhi was the major sublineage (82%) among the L3/CAS family (&chi;2=664, P=0.0001). Analysis showed that the majority of the clinical isolates with an unknown pattern had an evolutionary link with the L3/CAS strain, and nine (5.4%) of the unknown strains were epidemiologically linked and were tentatively named L3/CAS-KP (Khyber Pakhtunkhwa).Conclusion: The present study demonstrated that L3/CAS is the predominant lineage of MTB, widely distributed in different areas of the Khyber Pakhtunkhwa province of Pakistan. Spoligotyping patterns of some clinical isolates could not be matched to other reported patterns in an international database. Other tools, such as mycobacterial interspersed repetitive unit&ndash;variable number tandem repeat (MIRU-VNTR), will be helpful in future investigations into the epidemiological characteristics of clinical isolates in the Khyber Pakhtunkhwa province.Keywords: spoligotyping, VNTR, L3/CAS, genotyping, molecular characterization, genetic diversit

Novel Mutations in MPT64 Secretory Protein of Mycobacterium tuberculosis Complex

Tuberculosis (TB) is a global health problem caused by the Mycobacterium tuberculosis complex (MTBC). These bacteria secrete various proteins involved in the pathogenesis and persistence of MTBC. Among the secretory proteins, MPT64 (Rv1980C) is highly conserved and is also known as a major culture filtrate that is used in rapid diagnosis of MTBC. In the current study, we aimed to find the mutation in this highly conserved protein in isolates from the Pashtun-dominant province of Pakistan. We analyzed 470 M. tuberculosis whole-genome sequences of Khyber Pakhtunkhwa Province. Mutations in the MPT64 gene were screened through TB-Profiler and BioEdit software tools. The DynaMut web server was used to analyze the impact of the mutation on protein dynamics and stability. Among 470 MTB genomes, three non-synonymous mutations were detected in nine isolates, and one synonymous mutation (G208A) was found in four isolates. Mutation G211T (F159L), which was detected at the C-terminal domain of the protein in six isolates, was the most prominent. The second novel mutation, T480C (I70V), was detected in two isolates at the C-terminal side of the protein structure. The third novel mutation, A491C (L66R), was detected in a single isolate at the N-terminal side of the MPT64 protein. The effect of these three mutations was destabilizing on the protein structure. The molecular flexibility of the first two mutations increased, and the last one decreased. MPT64 is a highly conserved secretory protein, harboring only a few mutations. This study provides useful information for better managing the diagnosis of MTB isolates in high TB-burden countries

Novel mutations in MPT64 secretory protein of Mycobacterium tuberculosis complex

Tuberculosis (TB) is a global health problem caused by the Mycobacterium tuberculosis complex (MTBC). These bacteria secrete various proteins involved in the pathogenesis and persistence of MTBC. Among the secretory proteins, MPT64 (Rv1980C) is highly conserved and is also known as a major culture filtrate that is used in rapid diagnosis of MTBC. In the current study, we aimed to find the mutation in this highly conserved protein in isolates from the Pashtun-dominant province of Pakistan. We analyzed 470 M. tuberculosis whole-genome sequences of Khyber Pakhtunkhwa Province. Mutations in the MPT64 gene were screened through TB-Profiler and BioEdit software tools. The DynaMut web server was used to analyze the impact of the mutation on protein dynamics and stability. Among 470 MTB genomes, three non-synonymous mutations were detected in nine isolates, and one synonymous mutation (G208A) was found in four isolates. Mutation G211T (F159L), which was detected at the C-terminal domain of the protein in six isolates, was the most prominent. The second novel mutation, T480C (I70V), was detected in two isolates at the C-terminal side of the protein structure. The third novel mutation, A491C (L66R), was detected in a single isolate at the N-terminal side of the MPT64 protein. The effect of these three mutations was destabilizing on the protein structure. The molecular flexibility of the first two mutations increased, and the last one decreased. MPT64 is a highly conserved secretory protein, harboring only a few mutations. This study provides useful information for better managing the diagnosis of MTB isolates in high TB-burden countries

Novel mutations in genes of the IL-12/IFN-γ axis cause susceptibility to tuberculosis

Background: The IL-12/23/ISG15-IFN-γ pathway is the main immunological pathway for controlling intra-macrophagic microorganisms such as Mycobacteria, Salmonella, and Leishmania spp. Consequently, upon mutations in genes of the IL-12/23/ISG15-IFN-γ pathway cause increased susceptibility to intra-macrophagic pathogens, particularly to Mycobacteria. Therefore, the purpose of this study was to characterize the mutations in genes of the IL-12/23/ISG15-IFN-γ pathway in severe tuberculosis (TB) patients. Methods: Clinically suspected TB was initially confirmed in four patients (P) (P1, P2, P3, and P4) using the GeneXpert MTB/RIF and culturing techniques. The patients' Peripheral blood mononuclear cells (PBMCs) were then subjected to ELISA to measure Interleukin 12 (IL-12) and interferon gamma (IFN-γ). Flow cytometry was used to detect the surface expressions of IFN-γR1 and IFN-γR2 as well as IL-12Rβ1and IL-12Rβ2 on monocytes and T lymphocytes, respectively.The phosphorylation of signal transducer and activator of transcription 1(STAT1) on monocytes and STAT4 on T lymphocytes were also detected by flow cytometry. Sanger sequencing was used to identify mutations in the IL-12Rβ1, STAT1, NEMO, and CYBB genes. Results: P1's PBMCs exhibited reduced IFN-γ production, while P2's and P3's PBMCs exhibited impaired IL-12 induction. Low IL-12Rβ1 surface expression and reduced STAT4 phosphorylation were demonstrated by P1's T lymphocytes, while impaired STAT1 phosphorylation was detected in P2's monocytes. The impaired IκB-α degradation and abolished H2O2 production in monocytes and neutrophils of P3 and P4 were observed, respectively. Sanger sequencing revealed novel nonsense homozygous mutation: c.191 G>A/p.W64 * in exon 3 of the IL-12Rβ1 gene in P1, novel missense homozygous mutation: c.107 A>T/p.Q36L in exon 3 of the STAT1 gene in P2, missense hemizygous mutation:: c.950 A>C/p.Q317P in exon 8 of the NEMO gene in P3, and nonsense hemizygous mutation: c.868 C>T/p.R290X in exon 8 of CYBB gene in P4. Conclusion: Our findings broaden the clinical and genetic spectra associated with IL-12/23/ISG15-IFN-γ axis anomalies. Additionally, our data suggest that TB patients in Pakistan should be investigated for potential genetic defects due to high prevalence of parental consanguinity and increased incidence of TB in the country