Mycobacterium Tuberculosis Complex Research Articles

Rapid lateral flow test for Mycobacterium tuberculosis complex and non-tuberculous mycobacteria differentiation.

The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases. KEY POINTS: • Panel of mAbs for differentiating MTB versus NTM • IC strips for diagnosing MTBC and NTM after the BACTEC MGIT • Combined detection of MTP64 and Ag85B enhances diagnostic accuracy.

Decoding drug resistance in mycobacterium tuberculosis complex: genetic insights and future challenges.

Tuberculosis (TB), particularly its drug-resistant forms (MDR-TB and XDR-TB), continues to pose a significant global health challenge. Despite advances in treatment and diagnosis, the evolving nature of drug resistance in Mycobacterium tuberculosis (MTB) complicates TB eradication efforts. This review delves into the complexities of anti-TB drug resistance, its mechanisms, and implications on healthcare strategies globally. We explore the genetic underpinnings of resistance to both first-line and second-line anti-TB drugs, highlighting the role of mutations in key genes. The discussion extends to advanced diagnostic techniques, such as Whole-Genome Sequencing (WGS), CRISPR-based diagnostics and their impact on identifying and managing drug-resistant TB. Additionally, we discuss artificial intelligence applications, current treatment strategies, challenges in managing MDR-TB and XDR-TB, and the global disparities in TB treatment and control, translating to different therapeutic outcomes and have the potential to revolutionize our understanding and management of drug-resistant tuberculosis. The current landscape of anti-TB drug resistance demands an integrated approach combining advanced diagnostics, novel therapeutic strategies, and global collaborative efforts. Future research should focus on understanding polygenic resistance and developing personalized medicine approaches. Policymakers must prioritize equitable access to diagnosis and treatment, enhancing TB control strategies, and support ongoing research and augmented government funding to address this critical public health issue effectively.

Association of mutations in Mycobacterium tuberculosis complex (MTBC) respiration chain genes with hyper-transmission.

The respiratory chain plays a key role in the growth of Mycobacterium tuberculosis complex (MTBC). However, the exact regulatory mechanisms of this system still need to be elucidated, and only a few studies have investigated the impact of genetic mutations within the respiratory chain on MTBC transmission. This study aims to explore the impact of respiratory chain gene mutations on the global spread of MTBC. A total of 13,402 isolates of MTBC were included in this study. The majority of the isolates (n = 6,382, 47.62%) belonged to lineage 4, followed by lineage 2 (n = 5,123, 38.23%). Our findings revealed significant associations between Single Nucleotide Polymorphisms (SNPs) of specific genes and transmission clusters. These SNPs include Rv0087 (hycE, G178T), Rv1307 (atpH, C650T), Rv2195 (qcrA, G181C), Rv2196 (qcrB, G1250T), Rv3145 (nuoA, C35T), Rv3149 (nuoE, G121C), Rv3150 (nuoF, G700A), Rv3151 (nuoG, A1810G), Rv3152 (nuoH, G493A), and Rv3157 (nuoM, A1243G). Furthermore, our results showed that the SNPs of atpH C73G, atpA G271C, qcrA G181C, nuoJ G115A, nuoM G772A, and nuoN G1084T were positively correlated with cross-country transmission clades and cross-regional transmission clades. Our study uncovered an association between mutations in respiratory chain genes and the transmission of MTBC. This important finding provides new insights for future research and will help to further explore new mechanisms of MTBC pathogenicity. By uncovering this association, we gain a more complete understanding of the processes by which MTBC increases virulence and spread, providing potential targets and strategies for preventing and treating tuberculosis.

Detection of multidrug-resistance in Mycobacterium tuberculosis by phenotype- and molecular-based assays.

The whole-genome sequencing (WGS) is becoming an increasingly effective tool for rapid and accurate detection of drug resistance in Mycobacterium tuberculosis complex (MTBC). This approach, however, has still been poorly evaluated on strains from Central and Eastern European countries. The purpose of this study was to assess the performance of WGS against conventional drug susceptibility testing (DST) for the detection of multi-drug resistant (MDR) phenotypes among MTBC clinical strains from Poland and Lithuania. The study included 208 MTBC strains (130 MDR; 78 drug susceptible), recovered from as many tuberculosis patients in Lithuania and Poland between 2018 and 2021. Resistance to rifampicin (RIF) and isoniazid (INH) was assessed by Critical Concentration (CC) and Minimum Inhibitory Concentration (MIC) DST as well as molecular-based techniques, including line-probe assay (LPA) and WGS. The analysis of WGS results was performed using bioinformatic pipeline- and software-based tools. The results obtained with the CC DST were more congruent with those by LPA compared to pipeline-based WGS. Software-based tools showed excellent concordance with pipeline-based analysis in prediction of RIF/INH resistance. The RIF-resistant strains demonstrated a relatively homogenous MIC distribution with the mode at the highest tested MIC value. The most frequent RIF-resistance conferring mutation was rpoB S450L. The mode MIC for INH was two-fold higher among double katG and inhA mutants than among single katG mutants. The overall rate of discordant results between all methods was calculated at 5.3%. Three strains had discordant results by both genotypic methods (LPA and pipeline-based WGS), one strain by LPA only, three strains by MIC DST, two strains by both MIC DST and pipeline-based WGS, and the remaining two strains showed discordant results with all three methods, compared to CC DST. Considering MIC DST results, current CCs of the first-line anti-TB drugs might be inappropriately high and may need to be revised. Both molecular methods demonstrated 100% specificity, while pipeline-based WGS had slightly lower sensitivity for RIF and INH than LPA, compared to CC DST.

Comparative Genomic Analysis of Mycobacterium tuberculosis Isolates Circulating in North Santander, Colombia

Tuberculosis (TB) is an important infectious disease in relation to global public health and is caused species of the Mycobacterium tuberculosis complex (MTBC). In this study, we used whole-genome sequencing (WGS) and comparative genomics to investigate the genetic diversity of M. tuberculosis (Mtb) isolates circulating in North Santander (NS), Colombia. WGS was used for the phylogenetic and lineage characterization of 18 isolates of Mtb typed with orphan genotypes from 11 municipalities of NS between 2015 and 2018. The isolates studied were included in six sublineages from L4; the most frequent were 4.1.2.1, 4.3.3, and 4.3.4.2, corresponding to a proportion of 22.2%. The genome analysis conducted allowed the identification of a set of genetic variants mainly associated with determinants of virulence and evasion of the immune system (PPE34 and PE_PGRS2); adaptation and survival (PGL/p-HBAD); stress response (sigJ and sigM); geographic variability (PPE34); and carbohydrate and lipid metabolism (aldA, rocA, and cyp144). This is the first description of the molecular epidemiology of Mtb isolates circulating in NS achieved through WGS. It was possible to perform comparative genomics analyses between Mtb isolates against the universal reference H37Rv and Colombian UT205 genome, which can help us to understand the local genetic diversity and is relevant for epidemiological studies, providing insight into TB transmission dynamics in NS.

Identification of Mycobacterium tuberculosis complex species using dual-tube multiplex real-time PCR and determination of their rifampicin and isoniazid resistance profiles: A 5-year study

Identification of Mycobacterium tuberculosis complex species using dual-tube multiplex real-time PCR and determination of their rifampicin and isoniazid resistance profiles: A 5-year study

Characterization of a Novel Oxidative Stress Responsive Transcription Regulator in Mycobacterium bovis.

The antioxidant defense is critical for the survival of intracellular pathogens such as Mycobacterium tuberculosis complex (MTBC) species, including Mycobacterium bovis, which are often exposed to an oxidative environment caused by reactive oxygen species (ROS) in hosts. However, the signaling pathway in mycobacteria for sensing and responding to oxidative stress remains largely unclear. In this study, we characterize a TetR-type transcription regulator BCG_3893c, designated AotM, as a novel redox sensor in Mycobacterium bovis that increases mycobacterial tolerance to oxidative stress. AotM is required for the growth of M. bovis in the presence of 1 mM hydrogen peroxide. Loss of the aotM gene leads to altered transcriptional profiles with 352 genes significantly up-regulated and 25 genes significantly down-regulated. AotM recognizes a 14-bp palindrome sequence motif and negatively regulates the expression of a FAD-dependent oxidoreductase encoded by bcg_3892c. Overexpression of BCG_3892c increases intracellular ROS production and reduces the growth of M. bovis. In summary, we propose that AotM enhances the mycobacterial resistance against oxidative stress probably by inhibiting intracellular ROS production. Our findings reveal a novel underlying regulatory mechanism behind mycobacterial oxidative stress adaptation.

Single nucleotide variation catalog from clinical isolates mapped on tertiary and quaternary structures of ESX-1-related proteins reveals critical regions as putative Mtb therapeutic targets.

We mapped all non-synonymous single nucleotide polymorphisms onto each of the experimental and predicted ESX-1 proteins' structural models and inspected their placement. Varying sizes of conserved regions were found. Next, we analyzed predicted intrinsically disordered regions within our set of proteins, finding two putative long stretches that are fully conserved, and discussed their potential essential role in immunological recognition. Combined, our findings highlight new targets for interfering with Mycobacterium tuberculosis complex virulence.

Multidisciplinary Effort Leading to Effective Tuberculosis Community Outbreak Containment in Israel.

Tuberculosis (TB) is the second-most prevalent cause of mortality resulting from infectious diseases worldwide. It is caused by bacteria belonging to the Mycobacterium tuberculosis complex (MTBC). In Israel, TB incidence is low, acknowledged by the WHO as being in a pre-elimination phase. Most cases occur among immigrants from high TB incidence regions like the Horn of Africa and the former Soviet Union (FSU), with occasional outbreaks. The outbreak described in this report occurred between 2018 and 2024, increasing the incidence rate of TB in the region. Control of this outbreak posed challenges due to factors including a diverse population (including Ethiopian immigrants, Israeli-born citizens, and immigrants from other countries), economic and social barriers, and hesitancy to disclose information. The unique multidisciplinary team formed to address these challenges, involving the local TB clinic, district health ministry, health maintenance organization (HMO) infectious disease consultant, neighborhood clinic, and National Mycobacterium Reference Laboratory (NMRL), achieved effective treatment and containment. Whole genome sequencing (WGS) proved pivotal in unraveling patient connections during the outbreak. It pinpointed those patients overlooked in initial field investigations, established connections between patients across different health departments, and uncovered the existence of two distinct clusters with separate transmission chains within the same neighborhood. This study underscores collaborative efforts across sectors that successfully contained a challenging outbreak.

Prospective evaluation of thin-layer agar colour test in routine diagnosis of multidrug-resistant TB.

<sec><title>BACKGROUND</title>This study evaluated the diagnostic performance of the thin-layer agar MDR/XDR-TB Colour Test (CT), a Mycobacterium tuberculosis complex (MTBC) detection and direct drug susceptibility testing (DST) method with routine sputum, bronchoalveolar lavage and pleural fluid specimen.</sec><sec><title>METHODS</title>In a prospective study, the time and rate of MTBC detection were compared between CT, Löwenstein-Jensen, and MGIT media. Times until DST result, sensitivities, and specificities were evaluated between CT and MGIT 960 indirect DST.</sec><sec><title>RESULTS</title>The cultivation of 177 pulmonary specimens resulted in 83 MTBC-positive cultures. The sensitivity of CT for MTBC detection was 81.3% with a median time of 20 days compared to 13 days and 93.5% for MGIT. The sensitivity of CT for DST results was 100% for isoniazid and levofloxacin and 94.7% for rifampicin. The specificities for isoniazid and rifampicin DST were 97.3% and 98.0% for levofloxacin. The median time until a DST result was significantly shorter with CT than the BACTEC MGIT 960 system, 20 and 27 days, respectively, independent of the specimen type used.</sec><sec><title>CONCLUSIONS</title>The CT is a highly accurate and fast initial diagnostic test for high-incidence settings and could also be used as a first culture and direct DST in peripheral settings.</sec>.

Differential rates of Mycobacterium tuberculosis transmission associate with host-pathogen sympatry.

Several human-adapted Mycobacterium tuberculosis complex (Mtbc) lineages exhibit a restricted geographical distribution globally. These lineages are hypothesized to transmit more effectively among sympatric hosts, that is, those that share the same geographical area, though this is yet to be confirmed while controlling for exposure, social networks and disease risk after exposure. Using pathogen genomic and contact tracing data from 2,279 tuberculosis cases linked to 12,749 contacts from three low-incidence cities, we show that geographically restricted Mtbc lineages were less transmissible than lineages that have a widespread global distribution. Allopatric host-pathogen exposure, in which the restricted pathogen and host are from non-overlapping areas, had a 38% decrease in the odds of infection among contacts compared with sympatric exposures. We measure tenfold lower uptake of geographically restricted lineage 6 strains compared with widespread lineage 4 strains in allopatric macrophage infections. We conclude that Mtbc strain-human long-term coexistence has resulted in differential transmissibility of Mtbc lineages and that this differs by human population.

Viewpoint of multi-omics potential in tuberculosis research: identifying biomarkers for biomanufacturing of efficient control tools

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), remains a global health burden, claiming millions of lives annually. Despite the availability of a vaccine (the Bacillus Calmette-Guérin; BCG), diagnostics (smear microscopy and DNA-based diagnostics) and drugs for treatment, challenges like drug resistance, lower diagnostic sensitivity such as microscopy, instrumentation and high-level operational expertise requirement and poor vaccine efficacy pose a greater hinderance to TB monitoring and control. The inability of the BCG vaccine to protect against TB other than disseminated disease in neonates call for innovative approaches to provide improved interventions. This review highlights the potential of multi-omics as a powerful emerging tool offering a holistic insight into the intricate interplay between pathogens and the host immune response. It discusses the potential of multi-omics for discovering biomarkers as targets for rational drug design, production of more effective vaccines and as stable targets for production of universally applicable rapid diagnostics for early and accurate TB detection as well as monitoring treatment.

Detection of Mycobacterium tuberculosis complex in saliva by quantitative PCR: A potential alternative specimen for pulmonary tuberculosis diagnosis

BackgroundCurrent tuberculosis (TB) diagnostic tests primarily rely on sputum samples, yet many TB patients cannot produce sputum. This study explored whether saliva could be used instead of sputum to diagnose pulmonary TB (PTB). MethodThe study included 32 patients with confirmed PTB and 30 patients with other respiratory diseases (ORD). Saliva from all study participants was subjected to quantitative (qPCR) assays targeting the IS1081 gene for detection of M. tuberculosis complex DNA. ResultsThe sensitivity of saliva IS1081 qPCR was 65.6 % (95 % CI 48.4–80.2 %) with positive results for 21/32 PTB cases, while the specificity was 96.7 % (95 % CI 85.9–99.6 %) with negative results for 29/30 participants with ORD. Sensitivity improved to 72.4 % (95 % CI 54.6–86.0 %) when sputum-Xpert was used as the reference standard, while remaining similar at 65.5 % (95 % CI 47.4–80.7 %) when culture was used as the reference standard. In receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) for saliva IS1081 qPCR was 82.5 % (95 % CI 71.7–93.3 %). ConclusionSaliva testing offers a promising alternative to sputum for TB diagnosis among confirmed PTB cases. Larger multicenter studies, encompassing diverse clinical TB characteristics, are needed to provide improved estimates of diagnostic sensitivity and specificity.

Insights into mycobacteriome composition in Mycobacterium bovis-infected African buffalo (Syncerus caffer) tissue samples

Animal tuberculosis significantly challenges global health, agriculture, and wildlife conservation efforts. Mycobacterial cultures are resource-intensive, time-consuming, and challenged by heterogeneous populations. In this study, we employed a culture-independent approach, using targeted long-read-based next-generation sequencing (tNGS), to investigate the mycobacterial composition in 60 DNA samples extracted from Mycobacterium bovis infected culture-confirmed African buffalo tissue. We detected mycobacterial DNA in 93.3% of the samples and the sensitivity for detecting Mycobacterium tuberculosis complex (MTBC) was 91.7%, demonstrating a high concordance of our culture-independent tNGS approach with mycobacterial culture results. In five samples, we identified heterogenous mycobacterial populations with various non-tuberculous mycobacteria, including members of the Mycobacterium avium complex (MAC), M. smegmatis, and M. komaniense. The latter Mycobacterium species was described in South Africa from bovine nasal swabs and environmental samples from the Hluhluwe-iMfolozi Park, which was the origin of the buffalo samples in the present study. This finding suggests that exposure to environmental mycobacteria may confound detection of MTBC in wildlife. In conclusion, our approach represents a promising alternative to conventional methods for detecting mycobacterial DNA. This high-throughput technique enables rapid differentiation of heterogeneous mycobacterial populations, which will contribute valuable insights into the epidemiology, pathogenesis, and microbial synergy during mycobacterial infections.

Burden and factors associated with occupational tuberculosis infection among high-risk workers in Lahore District, Pakistan.

The main objective of the study was to estimate the burden of occupational tuberculosis infection in high-risk occupational workers and to identify risk factors associated with the prevalence of Mycobacterium tuberculosis complex (MTBC). An analytical cross-sectional study was conducted among high-risk occupational workers including veterinarians, abattoir workers, animal handlers, livestock farmers, and microbiology laboratory workers. Sputum samples were collected from 100 participants and polymerase chain reaction (PCR) tests were done to diagnose tuberculosis (TB) infection. Data on potential risk factors was collected in a pre-designed questionnaire. The MTBC prevalence ratio was estimated. Logistic regression analysis was conducted to identify risk factors and the crude odds ratio (OR) was calculated. Among the 100 enrolled high risk occupational workers, the prevalence of MTBC was 46% (95% CI: 35.98-56.25). Living in a joint family (OR 3.85, 95% CI: 1.58-9.37), and use of unpasteurized milk (OR 3.42, 95% CI: 1.4-8.39), were significantly associated with MTBC infection. Tuberculosis is a significant health burden in high-risk occupational groups, especially animal handlers and laboratory workers, in Lahore, Pakistan. The study also emphasized the need for formal work-related training, and enhanced zoonotic TB awareness among occupational workers.

New genetic biomarkers from transcriptome RNA-sequencing for Mycobacterium tuberculosis complex and Mycobacterium avium complex infections by bioinformatics analysis

The study aims to accurately identify differentially expressed genes (DEGs) and biological pathways in mycobacterial infections through bioinformatics for deeper disease understanding. Differentially expressed genes (DEGs) was explored by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Unique DEGs were submitted on least absolute shrinkage and selection operator (LASSO) regression analysis. 1,057 DEGs from two GSE datasets were identified, which were closely connected with NTM/ latent TB infection (LTBI)/active TB disease (ATB). It was demonstrated that these DEGs are mainly associated with detoxification processes, and virus and bacterial infections. Moreover, the METTL7B gene was the most informative marker for distinguishing LTBI and ATB with an area under the curve (AUC) of 0.983 (95%CI: 0.964 to 1). The significantly upregulated HBA1/2 genes were the most informative marker for distinguishing between individuals of IGRA-HC/NTM and LTBI (P < 0.001). Moreover, the upregulated HBD gene was also differ between IGRA-HC/NTM and ATB (P < 0.001). We have identified gene signatures associated with Mycobacterium infection in whole blood, which could be significant for understanding the molecular mechanisms and diagnosis of NTM, LTBI, or ATB.

Antimicrobial resistance profile and prevalence of Mycobacterium tuberculosis complex in Western Iran using spoligotyping method

Tuberculosis (TB) is a chronic infectious disease with multiple manifestations and gradual progression that remains a major health problem and a leading cause of death worldwide. In recent years, a number of DNA fingerprinting techniques have been developed to identify strains of the Mycobacterium tuberculosis (MTB) complex. Spoligotyping is one of the first PCR-based genotyping methods. Information on the number and identification of common strains among MTB complex samples in clinical samples from Kermanshah city is needed to develop more effective therapeutic strategies.This is a descriptive cross-sectional study of 41 sample patients with TB referred to Kermanshah Tuberculosis Centre between December 2021 and June 2022, including sputum, aspiration, urine, etc. First, the susceptibility of the developed bacteria to culture media was compared with that of isoniazid using the proportional method, and rifampin was determined according to the standard protocol. Demographic data of patients referred to the Centre for the Control of Lung Diseases were also recorded.In the next step, spoligotyping was carried out using the standard method and each strain pattern was recorded as an OCTAL code and compared with the information available at the World Bank on spoligotyping and its strains. Forty-one patients with pulmonary TB were tested using spoligotyping. Four MTB strains were identified, including H4, CAS, T1 and H1.The H4 strain also had the highest frequency with 16 samples (39%) among the MTB complex strains isolated using spoligotyping.The highest frequency of strains isolated using spoligotyping was associated with the H4 strain. It can be concluded that spoligotyping is very cost effective, simple, repeatable and highly sensitive.

Functional analysis of the Mycobacterium bovis AF2122/97 PhoPR system

The PhoPR system is a master regulator in Mycobacterium tuberculosis. A key difference between M. tuberculosis and Mycobacterium bovis is a G71I substitution in the M. bovis PhoR orthologue. Functional studies of the M. bovis PhoPR system have generated conflicting findings, with some research suggesting that the M. bovis PhoPR is defective while others indicate it is functional.We sought to revisit the functionality of the M. bovis PhoPR system. To address this, we constructed a phoPR mutant in the reference strain M. bovis AF2122/97. We employed a combination of growth assays and transcriptomics analyses to assess the phenotype of the mutant vs wild type and complemented strains. We found that the M. bovis AF2122/97 ΔphoPR mutant showed a growth defect on solid and liquid media compared to the wild type and complemented strains. The transcriptome of the M. bovis AF2122/97 ΔphoPR mutant was also altered as compared to wild type, including differential expression of genes involved in lipid metabolism and secretion. Our work provides further insight into the activity of PhoPR in M. bovis and underlines the importance of the PhoPR system as a master regulator of gene expression in the Mycobacterium tuberculosis complex.

Optimization of Mycobacterium tuberculosis DNA processing prior to whole genome sequencing

The process of whole genome sequencing of the Mycobacterium tuberculosis complex is dependent on complete the inactivation of the strain and subsequent DNA extraction. The objective of this study was to optimise the two steps.Firstly, the efficacy of Triton X-100 as a solvent for the inactivation step was evaluated. This solvent has been demonstrated to be effective in killing bacteria and is less toxic than the previously employed chloroform. For the extraction step, two lysis methods were evaluated: enzymatic (B1 protocol) and mechanical (B2 protocol). For whole genome sequencing, the Nextera XT DNA library preparation protocol was performed for both the B1 and B2 protocols. Subsequently, each library was subjected to whole-genome sequencing. The results demonstrated that heat lysis inactivation with Triton was effective, with no bacteria remaining viable following this treatment. The enzymatic and mechanical extraction protocols yielded comparable results in terms of DNA quantity and quality. The sequencing results showed that there was no significant difference in read depths between the two protocols. In conclusion, for MTBC strains, we recommend the use of our Triton inactivation method, which meets biosafety expectations.

Non-tuberculous mycobacterium isolations from tuberculosis presumptive cases at the National Tuberculosis Reference Laboratory Kenya, 2018–2019

Background: Mycobacterial pathogens are among the top causes of diseases in humans. In Kenya, incidences of Non-Tuberculous Mycobacteria (NTM) species have steadily been on the increase. Most NTMare resistant to first line treatment of tuberculosis and have a challenge in timely and accurate diagnosis. Misdiagnosis has led to prescribing anti-tuberculosis regimens to patients suffering from NTM. We aimed to determine the most prevalent Non-Tuberculous Mycobacterium in Kenya. Methods: We reviewed records from the National Tuberculosis Reference Laboratory(NTRL ) Laboratory information management system (LIMS) between January 2018 and December 2019 for the patients on surveillance. All isolates were cultured in Mycobacterial Growth Indicator Tubes (MGIT) and incubated for detection using BACTEC™ MGIT™ system. Those with negative acid-fast bacilli (AFB) growth and negative for Mycobacterium Tuberculosis Complex Species (MTBC) protein-MPT64 were suggestive of NTM infections, which were sub-cultured in MGIT and characterized using Line Probe Assay (LPA) GenoType® MTBDR CM/AS. Descriptive and bivariate analysis was done. Results: Of the total 24,549 records reviewed, 167(0.7%) were NTM isolates. Males comprised of 74.2% (124/167), and the mean age was 42 years (SD±16), age group 35-44 years had the highest NTM at 26.3% (44/167). Nairobi had 12.6% (21/167), Mombasa 10.8% (18/167), Kilifi and Meru each had 7.8% (13/167). Eleven isolated species comprised of Mycobacterium intracellulare 35.3% (65/167), M. fortuitum at 27% (48/167), and M. scrofulaceum at 10.2% (17/167). Previously treated patients had higher NTM [63.5% (106/167)] than Drug-resistant follow-up patients [26.9% (45/167)]. Coinfection with HIV was at 27.5% (46/167). Conclusion: Previously treated patients should have an additional screening of NTMS, and drug susceptibility testing should be done before initiation of treatment.