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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 1  |  Page : 60-69

Genetic diversity of nontuberculous mycobacteria among symptomatic tuberculosis negative patients in Kenya


1 School of Health Sciences, Meru University of Science and Technology; University of Nairobi Institute of Tropical and Infectious Diseases, Nairobi, Kenya
2 National Tuberculosis Reference laboratory, Kenya Medical Research Institute (KEMRI), Kenya
3 School of Health Sciences, Meru University of Science and Technology, Nairobi; Centre for Molecular Biosciences and Genomics, Kenya
4 Kenya Medical Research Institute (KEMRI), Kenya

Date of Submission02-Nov-2021
Date of Decision20-Nov-2021
Date of Acceptance29-Dec-2021
Date of Web Publication12-Mar-2022

Correspondence Address:
Zakayo Maingi Mwangi
Meru University of Science and Technology, P O Box 972-60200, Meru
Kenya
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_224_21

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  Abstract 


Background: Non-Tuberculous Mycobacteria (NTM) transmission to humans occurs through inhalation of dust particles or vaporized water containing NTM leading to pulmonary manifestations. NTM infections are often misdiagnosed for tuberculosis (TB) due to their similar clinical and radiological manifestations. Aims and Objectives: We, therefore, performed a species-level identification of NTM in symptomatic TB negative patients through sequencing of the hsp65 gene. Materials and Methods: We conducted a cross-sectional study at the National Tuberculosis Reference Laboratory in the period between January to November 2020. One hundred and sixty-six mycobacterial culture-positive samples that tested negative for TB using capilia underwent Polymerase Chain Reaction targeting the hsp65 gene. Isolates showing a band with gel electrophoresis at 441 bp position were sequenced using Sanger technology. Geneious software was used to analyze the obtained sequences, and the National Center for Biotechnology Information gene database identified NTM species for each isolate. A phylogenetic tree was constructed from the DNA sequences and evolutionary distances computed using the general time-reversible method. Pearson chi-square was used to determine the association between NTM infection and participants' characteristics. Results: Our study identified 43 different NTM species. The dominant NTM belonged to Mycobacterium avium complex 37 (31%). Slow-growing NTM were the majority at 86 (71%) while rapid-growing NTM were 36 (29%). A significant association (P<0.05) was observed for regions and age, while patient type had a weak likelihood of NTM infection. Conclusion: Our study characterized the diversity of NTM in Kenya for the first time and showed that species belonging to M. Avium Complex are the most prevalent in the country.

Keywords: hsp65, nontuberculous mycobacteria genetic diversity, nontuberculous mycobacteria, rapid-growing nontuberculous mycobacteria, slow-growing nontuberculous mycobacteria


How to cite this article:
Mwangi ZM, Mukiri NN, Onyambu FG, Wallace BD. Genetic diversity of nontuberculous mycobacteria among symptomatic tuberculosis negative patients in Kenya. Int J Mycobacteriol 2022;11:60-9

How to cite this URL:
Mwangi ZM, Mukiri NN, Onyambu FG, Wallace BD. Genetic diversity of nontuberculous mycobacteria among symptomatic tuberculosis negative patients in Kenya. Int J Mycobacteriol [serial online] 2022 [cited 2022 May 21];11:60-9. Available from: https://www.ijmyco.org/text.asp?2022/11/1/60/339505




  Introduction Top


Nontuberculous mycobacteria (NTM) are ubiquitous environmental organisms that are present in habitats such as water, soil, and dust. While in the environment, NTM colonies coalesce in clusters forming biofilms that enable their persistence in adverse conditions such as harsh weather and degradation by disinfectants.[1],[2] The hydrophobicity of the biofilms enhances NTM dispersion and subsequent transmission to humans mainly through inhalation of dust particles or vaporized water containing NTM leading to NTM pulmonary disease (NTM-PD),[3],[4] but there is no evidence of human to human transmission.[5] Environmental and climatic conditions influence the distribution of more than the 200 NTM species[6] with specific species predominating various geographic regions.[7] For instance, Mycobacterium avium Scientific Name Search  complex (MAC), Mycobacterium gordonae and Mycobacterium xenopi were dominant in the different geographical regions within Europe,[8] in the Middle East Mycobacterium abscessus, Mycobacterium fortuitum, and Mycobacterium intracellulare were commonly isolated,[9] M. fortuitum was the most common NTM in India.[10] MAC was dominant in Singapore and Japan[10] while MAC, M. intracellulare, and Mycobacterium kansasii were the most common in Sub-Saharan Africa.[11]

The NTM-PD clinical presentation is similar to that of Mycobacteria tuberculosis (MTB) and is often misdiagnosed for MTB.[12] This leads to treatment complications since MTB and NTM management strategies are incongruent.[13] The identification of NTM is important in the determination of the clinically relevant species and deciding on the appropriate treatment regimen for the infection since mycobacterium susceptibility to antimicrobial agents varies for individual species.[13],[14],[15],[16] Classical NTM identification and species differentiation procedures are simple and cost-effective and utilize phenotypic characteristics such as their growth rate, pigment production, growth at different temperatures, and biochemical analysis including niacin accumulation test, nitrate reduction test, urea hydrolysis among several others.[17] These methods however have limitations in NTM diagnosis due to their time-consuming, tedious nature, and lack of accurate distinction of closely related species.[18],[19],[20]

Molecular methods are sufficient and highly efficient in the discrimination of NTM species in a fast and reliable manner a property that phenotypic methods lack.[6],[21],[22] The most commonly used molecular technique is by targeted sequencing of common housekeeping genes including 16s rRNA, rpoB, and hsp65. The sequences of these genes exhibit immense discriminatory power in their identification of the NTM species, hence providing a robust phylogenetic tree that enables a proper classification of NTM to either rapid or slow growers, and gives a better understanding of their evolutionary diversity.[23] Further, concatenation of the sequences belonging to these genes enables the description and characterization of novel NTM, hence increasing the number of identifiable NTM species in the gene databases.[24] Line probe assays (LPA) is a molecular technique that has gained popularity in reference microbiology laboratories within resource-limited settings.[25] The assay employs the reverse hybridization technique where amplicons are hybridized on a nitrocellulose membrane strip and result interpretation is done based on the presence of bands at various points on the strip enabling for the simultaneous detection and identification of NTM species.[18],[26] The commonly used LPA is GenoType® Mycobacterium CM/AS assay (Hain Lifescience, Nehren Germany) which is a commercial kit that relies on partial amplification of the 23S rRNA.

The 16s rRNA is composed of highly conserved as well as variable regions making it the commonly studied gene for bacterial identification and a resourceful target for studying phylogenetic relationships.[19] This gene however has a high level of inter-species sequence similarity with some mycobacteria having absolute identical homology in their sequences. For instance, M. abscessus/Mycobacterium bolletii/Mycobacterium massiliense, ycobacterium austroafricanum/Mycobacterium vanbaalenii, M. kansasii/Mycobacterium gastri, Mycobacterium senegalense/Mycobacterium houstonense and, Mycobacterium mucogenicum/Mycobacterium phocaicum present with 0% inter-species divergence making the 16s rRNA gene less suitable for mycobacteria species differentiation.[27] The rpoB gene encodes the β subunit of the RNA polymerase, an enzyme responsible for RNA synthesis.[28] This gene has a sensitivity of 92.71% to 100% for mycobacteria species identification compared to that of 16s rRNA that has a 96.57% to 100% sensitivity.[27],[29],[30] The hsp65 gene belongs to the heat shock protein family that is involved in intracellular protein folding, assembly, and transport thus being highly immunogenic. Compared to 16s rRNA and rpoB, the hsp65 gene sequence is more diverse with a high genetic heterogeneity and a lower inter-species similarity percentage ranging from 89.2% to 100%, therefore producing a more robust phylogenetic tree with most nodes having bootstrap P value above 80%.[18],[23],[27],[31],[32]

The application of the above-mentioned molecular diagnostic techniques that detect and distinguish NTM species have played a major role in the increased reporting of NTM cases especially in Sub-Saharan Africa.[11] The detection of NTM in Kenya has relied on LPA which is limited to identifying NTM complexes with the GenoType Mycobacteria CM/AS recognizing 15 and 16 NTM species respectively, hence limiting a comprehensive overview on NTM species diversity.[18],[33]

In this study, we obtained sputum samples from symptomatic tuberculosis (TB) culture-negative patients and identified the NTM species through sequencing of the hsp65 gene, and also described their evolutionary diversity. NTM species results identified by hsp65 sequencing were also compared to those identified using line probe assay (GenoType Mycobacteria CM/AS).


  Methods Top


Samples

Sputum samples received at the National Tuberculosis Reference Laboratory (NTRL) underwent mycobacterial culture and identification procedures. A total of 165 NTM samples received from January to November 2020 were aliquoted and transferred to the Molecular and Infectious Diseases Research Laboratory for sub-culturing and hsp65 polymerase chain reaction (PCR).

Laboratory procedures.

Sample processing, mycobacterial culture, and growth identification

The sputum samples were decontaminated using the N-acetyl-L-cysteine 2% NaOH (NALC-NaOH) procedure, then inoculated into mycobacteria growth indicator tube (MGIT) and Lowenstein-Jensen (LJ) media, incubated at 37°C and monitored for growth for up to 6 and 8 weeks respectively. At the same time, sputum smears were prepared, air dried, heat-fixed then fluorochrome stained with auramine O where mycobacteria appeared as bright yellow fluorescent rods when viewed under a light-emitting diodes microscope.

The culture growth in MGIT and LJ underwent the Mtb identification testing using the standard deviation Bioline TB Ag MPT64 assay (capilia) (Standard Diagnostics, Yongin-si, Gyeonggi-do, Republic of Korea), and capilia positive samples were excluded from the study. The capilia negative samples underwent ZN microscopy with the presence of AFB indicating a possible NTM.

DNA extraction

Mycobacterial DNA was extracted from 500 μL of re-suspended colonies using GenoLyse® (Hain Lifescience, Nehren, Germany) according to the manufacturer's instructions. Briefly, 100ul of lysis buffer (A-LYS) buffer was added to each cryovial containing the resuspended colonies and incubated for 5 min at 95°C after which 100ul neutralization buffer (A-NB) was added and centrifugation was done at 5000G for 10 min. The supernatant was transferred to a newly labeled cryovial awaiting PCR.

Conventional polymerase chain reaction, gel electrophoresis, and DNA purification

A conventional PCR targeting the hsp65 gene was conducted using the GoTaq® Green Master Mix (Promega, Madison, Wisconsin, USA) in a final reaction volume of 13 μl comprising 6.25 μl of 2X GoTaq Hot Start Green Master Mix, 2.5 μl DNA template, 0.25 μl of each of both F-(5′-ACCAACGATGGTGTGTCCAT-3′) and R-(5′-CTTGTCGAACCGCATACCCT-3′) primers at a final concentration of 10 pmoles, and 3.75 μl of nuclease-free water to make up the reaction volume. Thermal cycling conditions were 1 cycle of 94°C for 4 min, 35 cycles of 94°C for 1 min, 57°C for 1 min, 72°C for 1 min and a final extension for 10 min at 72°C. Amplified products were confirmed on a 1% Agarose gel stained with 4.6 μl SYBR safe DNA stain (Invitrogen, Carlsbad, California, USA), and bands at 441 bp were observed in an Ultra Violet gel viewer. The PCR products were enzymatically purified using ExoSAP IT (Applied Biosystems, Foster City, California, USA). Purification conditions were 37°c for 15 min followed by a second incubation at 80°c for 15 min and a final cooling step at 4°C for 5 min.

Hsp65 sequencing

The purified amplicons were sequenced in the forward direction by Sanger sequencing using Big Dye™ Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) and the forward primer. The sequencing reaction was a 10 μl reaction comprising 1.25 μl of Big Dye Terminator, 3 μl of × 5 Sequencing Buffer, 1 μl of 1 pmol of the sequencing primer, and 1.5 μl of the PCR product. The reaction volume was made up by adding 3.25 μl of nuclease-free water. The reaction proceeded through 96°C for 1 min then 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min.

Purification of cycle-sequencing products was done using the BigDye X Terminator™ purification kit following manufacturer's instructions (Applied Biosystems, Foster City, California, USA) and purified products were loaded onto the ABI 3730 genetic analyzer (Applied Biosystems, Foster City, California, USA) for capillary electrophoresis.

Data analysis

Geneious software was used to align raw AB1 files from the ABI 3730 then sequences for each sample were compared with those in the GenBank (National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/) and (hsp65)-BLAST (hsp65-BLAST) (http://hsp65blast.phsa.ca/blast/blast. html) DNA sequence database. NTM species identification was confirmed if a 97% match was achieved.

Statistical data analyses were performed using STATA version 13 (Statacorp LLC, Texas, USA). The Pearson's Chi-square test was used to compare differences in proportions; variables with P < 0.05 were considered statistically significant.

Phylogenetic analysis

Sequences of the hsp65 were trimmed to start and end at the same nucleotide position for all isolates. The alignment of multiple sequences was conducted with Geneious prime 2020.2 software then MEGA X version 10.2.4 software performed phylogenetic analysis on the 440 bp sequence.

The phylogenetic tree was constructed from the DNA sequences by using the neighbor-joining method, and the evolutionary distances were computed using the general time-reversible method. The gene sequence for M. tuberculosis H37Rv was used as the phylogenetic root.


  Results Top


Polymerase chain reaction of the hsp65 gene was performed to all the 166 samples and a DNA band at approximately 441 bp was observed with agarose gel for 146 (88.5%) isolates. Twenty (11.5%) isolates did not show a band during gel electrophoresis (C9, C22, C37, C38, C45, C50, C51, C55, C63, C73, C85, C97, C105, C112, C113, C114, C120, C135, C149, and C161) and were excluded from further analysis. Sequence analysis of the hsp65 for the 146 PCR products showed NTM species in 122 (84%) of the isolates.

A total of 50 different species were identified with NTM comprising 43 (86%), and 7 (14%) were found to be organisms other than NTM. The non-NTM species observed include M. tuberculosis, Rothia spp, Norcadia spp, Kocuria spp, Streptomyces spp, Rhodococcus spp, and Planctomycetes spp.

The frequently isolated NTM belonged to M. avium complex (31%) (M. avium subspecies, M. intracellulare, Mycobacterium colombiense, Mycobacterium yongonese, Mycobacterium parascrofulaceum, Mycobacterium paratuberculosis, and Mycobacterium timonense). Second most isolated NTM species belonged to M. fortuitum complex (20%) (M. fortuitum subsp fortuitum, Mycobacteriumconceptionense, M. senegalense, Mycobacterium peregrinum, Mycobacterium mageritense, Mycobacterium neworleansense, and Mycobacterium porcinum), followed by M. abscessus complex species (14%) (M. abscessus subsp. bollettii and M. abscessus subsp. abscessus). The species belonging to these three complexes accounted for 65% of all mycobacteria identified.

Most samples came from patients living in the Coastal region (41%), followed by Nairobi (26%) region. The Coastal region had the highest (18%) variety of NTM species with M. fortuitum (44%) being the most common NTM. The median age of participants was 39 years (interquartile range, 20–50) with the majority belonging to the age group between 30 and 39 years. The majority of subjects with NTM were male (73%). Patients on treatment follow-up for multi-drug resistant TB (MDR-TB) (31%) were the majority [Table 1].
Table 1: Sociodemographic characteristics of patients with nontuberculous mycobacteria

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To investigate the genetic relatedness of the isolated NTM, a phylogenetic tree was constructed. Phylogenetic analysis based on the hsp65 revealed very close genetic similarity in the NTM species, with clear separation of the slowly and rapidly growing mycobacteria. Slow-growing NTM were the majority at 86 (71%) [Table 2] while rapid-growing NTM were 36 (29%) [Table 3]. Each mycobacterial species was identified as a distinct entity in the phylogenetic tree [Figure 1].
Table 2: Frequency of slow-growing nontuberculous mycobacteria and their phylogenetic complexes

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Table 3: Frequency of rapidly growing nontuberculous mycobacteria and their phylogenetic complexes

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Figure 1: Phylogenetic tree showing the diversity of nontuberculous mycobacteria species isolated from sputum samples in symptomatic tuberculosis negative patients in Kenya. The tree was rooted to mycobacterium tuberculosis H37Rv which was also used as a positive control in the analysis

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The results of NTM identified through hsp65 gene sequencing and GenoType Mycobacterium CM/AS (Hain Lifesciences, Nehren, Germany) were compared for the 122 isolates. Eighty-two (67%) were identical in speciation of NTM [Table 4] while 24 (19%) were discordant for both assays [Table 5]. The GenoType Mycobacterium CM/AS assay was unable to identify 16 (13%) NTM species that were later on identified by hsp65 gene sequencing [Table 6].
Table 4: Similar results for nontuberculous mycobacteria speciation by genotype mycobacterium common mycobacteria/additional species and hsp65 gene sequencing

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Table 5: Discordant results in nontuberculous mycobacteria identification by genotype mycobacterium common mycobacteria/additional species and hsp65 partial sequencing

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Table 6: Nontuberculous mycobacteria identified by hsp65 but not identifiable by genotype mycobacteria common mycobacteria/additional species

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  Discussion Top


Global estimates show that MAC is the dominant NTM comprising between 34% and 61% of NTM isolated across the different continents.[34],[35] These findings are consistent with our study that identified species belonging to MAC (31%) (M. avium subspecies (32%), M. intracellulare (27%), M. yongonese (18%), M. parascrofulaceum (5%), M. paratuberculosis (2%), and M. timonense (2%)) as the most widely isolated NTM in Kenya. Other countries with similar findings include China,[36],[37],[38] Russia,[39] America.[40],[41] various countries in Europe[42] and Africa.[11],[19],[43],[44],[45] The high infectivity rate of MAC species could be attributed to their seemingly abundant nature and distribution in different environmental sources such as water and soil, consequently increasing its ease of spread and infection to humans.[19],[46] MAC has been highly associated with NTM-PD;[47] however, it remains unclear how its infectivity relates to NTM-PD.[48]

NTM species distribution varies in different geographical locations with environmental conditions contributing towards their distribution patterns.[8] Our findings suggest a regional variation in the diversity of NTM and a possible influence by the different geographical or environmental landscapes within Kenya.[11],[32] For instance, Nairobi is warm and wet with M. senegalense (15%) being the dominant NTM, Coastal region is very hot and very wet with a majority of the isolates having M. fortuitum (44%), M. abscessus bolletii (19%) was the most common in the Mt. Kenya region which is cold and wet, North Rift Valley is hot and dry and had M. yongonense (33%) as the prevalent NTM, and the Western region is hot and wet with M. avium (33%) as the most common NTM species. These findings may be inconclusive due to the limited number of samples analyzed in our study, and a need for larger and more systematic studies capturing both human and environmental information is required for a more comprehensive understanding of environmental NTM species distribution.[8] These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location.[49]

Certain demographic characteristics such as age and gender have been shown to increase the likelihood of NTM infection.[49],[50] In our study, persons with pulmonary NTM infection were mostly in the youthful age group (30–39 years) and these findings are similar to those of other resource-limited countries.[11] NTM was less likely to infect females suggesting a likelihood of estrogen conferring a protective effect by up-regulating immunological response to clearing of bacterial infections.[51],[52]

Pulmonary NTM infection was found to be commonly associated with patients on follow-up for MDR-TB (31%). MDR-TB patients in Zambia and Nigeria recorded similar findings with NTM prevalence of 30% and 56% respectively.[50] Sub-Saharan African countries have the greatest burden of TB infections.[53] Pulmonary TB infections compromise the structural integrity of the lungs subsequently providing a suitable niche for NTM adherence and multiplication, hence the high number of NTM observed in MDR-TB cases.[54] A co-infection of TB and NTM could also be a possible occurrence in MTB-DR cases where the presence of NTM is attributed to its failure to respond to the first-line anti-TB drugs.[55]

The partial sequencing of the hsp65 gene was seen to be more sensitive in its ability to discriminate NTM to the species and subspecies level compared to GenoType Mycobacterium CM/AS (Hain Lifesciences GmbH, Nehren, Germany).[56] A worthwhile observation was that 10 (71%) of the 14 M. avium detected by hsp65 had been identified as M. intracellulare using Genotype Mycobacterium CM/AS (Hain Lifesciences, Nehren, Germany) suggesting a limited intraspecies detection heterogeneity by Genotype Mycobacterium CM/AS.[57] These occurrences for the Genotype Mycobacterium CM/AS could be attributed to its target gene the 23S rRNA having more conserved and few hypervariable regions, hence limiting its capacity to differentiate closely related NTM.[57] The contrasting results observed between hsp65 sequencing and Mycobacterium CM/AS in NTM species identification could either be due to mislabeling of sequences in the genetic databases, or a possible co-infection of two different NTM.[57]


  Conclusion Top


Our study characterized the diversity of NTM in Kenya for the first time and identified 43 different NTM species. MAC was the most prevalent in the country followed by M. fortuitum complex and M. abcessus complex species.

Slow-growing NTM were the majority at 86 (71%) while rapid-growing NTM were 36 (29%). A significant association (P < 0.05) was observed for regions and age, while patient type had a week likelihood of NTM infection.

Limitation of study

Our study lacked clinical details on the participants, such as underlying lung conditions and HIV status, making it difficult to identify clinically relevant NTM and their association with NTM-PD. Despite these limitations, our study characterized the diversity of NTM in Kenya for the first time and identified that species from the M. avium complex are the most prevalent in the country. Furthermore, we have shown that the diversity of mycobacteria species is high in Kenya, implying that before treatment of suspected TB patients, doctors should consider deeper characterization beyond just positivity, as this would provide a better guide to the appropriate treatment rather than assuming and treating for TB in all of these cases.

Ethical clearance

This study was approved by Kenyatta National Hospital-University of Nairobi Ethics Review Committee (Ref: KNH-ERC/A/38) on January 30, 2020. Waiver for individual informed consent was granted as the study utilized remnant clinical samples and the research posed no greater than minimal risk to the study subjects.

Acknowledgment

We are grateful to the management of the National Public Health Laboratories- Kenya for granting us permission to access the NTRL and carry out this research. Much appreciation to the laboratory staff at the National Tuberculosis Reference.

Laboratory for their technical support during the collection and initial analysis of sputum samples for this study.

Financial support and sponsorship

This work was supported by the Meru University of Science and Technology 6th call for proposals awarded to Zakayo Maingi Mwangi. The content is solely the responsibility of the authors and does not represent the views of the funders.

Conflicts of interest

There are no conflicts of interest.



 
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