Characterization of Non-Tuberculous Mycobacteria Pathogenic Species in Mali Using Genotype Mycobacterium Cm/As and 16s Sequencing during Tuberculosis Diagnosis or Treatment Follow-Up

Abstract

Introduction: Non-Tuberculous Mycobacteria (NTM) are environmental mycobacteria, some of which can cause pulmonary or extrapulmonary disease. We aimed to characterize species identified in our laboratory from suspected samples between 2009 and 2023.
Methods: We conducted a cross-sectional study between 2009 and 2023 at the National Institute of Public Health in Mali using clinical specimens positive for Acid-Fast Bacilli (AFB) in microscopy and negative by Xpert MTB/RIF and positive culture which were negative to MPT64 protein. The 16S nested PCR, was performed at the Institute of Tropical Medicine (ITM) in Antwerp, Belgium followed by Sanger sequencing at BaseClear (BE Leiden, The Netherlands) for 16S-positive amplicons. The Line Probe Assays (LPAs) GenoType Mycobacterium CM and AS VER 2.0 were realized from liquid or solid cultures at the National Reference Laboratory at the National Institute of Public Health in Mali.
Results: Clinical samples from 155 patients suspected of TB or drug resistance TB naive of treatment or during monitoring including 80 clinical samples and 75 positive cultures. Of these, 122 were NTM-positive results of which 47 were clinical samples and 75 cultures. 16S-PCR was positive for 47 (59%) of the 80 clinical samples and 71 (98,6%) of the 72 cultures. Sequencing was performed on 118 positive amplicons and LPA on 51 cultures. M. Avium Complex (MAC) was detected in nearly half (47%; 57/122), followed by M. simiae (13.1%), M. fortuitum (7.4%) and M. abscessus (4.9%).
Conclusion: A significant proportion of NTM identified in our patients, during TB diagnosis or treatment monitoring were potentially pathogenic species. The introduction and use of molecular platforms enabling differential detection of mycobacterial species will provide an opportunity for better management of TB and NTM diseases.

Keywords:GeneXpert, LPA, NTM, TB, Sequencing, 16S gene

Introduction

Non-Tuberculous Mycobacteria (NTM), also named atypical mycobacteria or mycobacteria other than Mycobacterium Tuberculosis Complex (MTBC) and Mycobacterium leprae, are environmental mycobacteria that can infect humans and animals [1]. Despite being ubiquitous, some of them are officially recognized as responsible for pulmonary or extrapulmonary diseases [2]. Although NTM and MTBC are different species, they all appear as Acid-Fast Bacilli (AFB) on microscopy. This property can lead to misdiagnosis, particularly in countries with limited resources where rapid molecular diagnostic tests or culture are not routinely available for all patients. In a previous study in our laboratory, 8.6% of AFB-positive patients were negative to Xpert MTB/RIF and 3.4% were carrying an NTM [3]. Unlike tuberculosis, there is no standardized treatment regimen against NTM diseases. They are treated with commonly used antibiotics combined with some antibiotics used against tuberculosis [1]. Before initiating treatment, the disease must be established based on clinical, radiological and microbiological criteria, including identification of the species to establish its pathogenicity [2]. The use of molecular methods, which are more specific than microscopy and faster than culture, has improved the rate of detection and identification of mycobacteria species. The 16S ribosomal RNA gene, present in all micro-organisms, is highly conserved and evolves slowly, making it a unique target that is widely used for phylogenetic studies of prokaryotes [4]. The 23S gene is also used to characterize mycobacteria, for example in LPAs [5]. Currently, more than 200 NTM species are officially recognized [6], with only a few, identified as pathogenic for humans [2]. Mycobacterium Avium Complex (MAC) sub-species are the most frequently detected across various geographic areas [7-16] and along with the generally non-pathogenic M. terrae complex are the best-studied NTM species [17]. Other important species of NTM are M. abscessus, M. celatum, M. fortuitum, M. kansasii, M. marinum, M. simiae and M. smegmatis [17]. In this study, we characterized the NTM species detected from 2009 to 2023 at the National Reference Laboratory for Tuberculosis (NRL) in Bamako, Mali, using the GenoType Mycobacterium CM/AS and Sanger sequencing of the 16S gene.

Materials and Methods

Study Design

A cross-sectional study was designed to characterize NTM species detected at the NRL from samples collected between 2009 and 2023. We included patients of any age who were referred by TB diagnostic and treatment centres across the country for TB diagnosis, or suspicion of drug-resistant TB during TB treatment monitoring. Socio-demographic and clinical data were collected from the laboratory registers and medical records. Species were identified from clinical specimens (sputum, gastric fluid, pleural fluid, and pus), or from liquid or solid culture, either freshly isolated (n=45) or retrieved from the laboratory’s strain bank at -80°C (n=30). For this study, we selected clinical specimens that were positive at microscopy and negative for GeneXpert MTB/RIF, and cultures that were negative by the MPT64 (75) assay

Identification by Sanger Sequencing Method

The nested 16S-PCR, was performed at the Institute of Tropical Medicine (ITM) in Antwerp, Belgium, using the primers depicted in table 1, for the 72 out of 75 cultures and all the 80 clinical specimens (microscopy+/Xpert negative) collected during the first round of the national drug resistance TB survey (Table 1).

Table 1:Primers for 16S nested PCR to detect NTM.

Sanger sequencing was performed at BaseClear (BE Leiden, The Netherlands), using the 16S- positive amplicons. The obtained sequences were uploaded in the Basic Local Alignment Search Tool (BLAST) of the National Centre for Biotechnology Information (NCBI) for comparison to reference strain sequences.

LPA Identification

The GenoType Mycobacterium CM/AS VER 2.0 was performed at the NRL using liquid or solid cultures according to the manufacturer’s recommendations [18]. Prior to the amplification, DNA was extracted from colonies by heat shock (95°C for 20 minutes, followed by 10 minutes in an ice bath). When the species could not be identified with the GenoType CM kit, we repeated the hybridization with the GenoType Mycobacterium AS [19].

Data Analysis

The data was recorded in Excel (Version 2020) and the percentages were calculated by crosstab, while p-values and odds ratios with 95% confidence intervals were generated using analysis in IBM SPSS Statistics.

Ethical Issues

Information on the patient’s name and address was removed from the data and each patient was identified by a unique identification number. Informed consent was not required from patients seen routinely in the laboratory outside studies, yet patients were informed that other tests, in addition to those requested by the prescriber, would be performed to identify the detected bacilli. The study was approved by the Ethics committee of the National Institute of Research in Public Health under the number N° 08/2018/ CE-INRSP.

Results

Detection and Characterization of NTM Species

In total, sample of 155 different patients were tested including 80 clinical samples and 75 positive cultures including 30 from laboratory biobank. Of these, 125 were AFB positive and Xpert/MTB not detected and in which, NTM species were detected in 92 (73,6%) (Figure 1). A total of 122 patients were carriers of NTM species, 47 from clinical samples including 2 extrapulmonary (pus and pleural fluid) and 120 pulmonary (117 sputum and 3 gastric fluids), 45 from fresh cultures and 30 from laboratory biobank. Among these patients, 54 (44.3%) were previously treated for TB and 68 (55.7%) were newly diagnosed patients.

Note*:AFB=acid-fast bacilli, AS=Additional Species, CM=Common Mycobacteria, n=number; NTM=non-tuberculous mycobacteria, MTB=Mycobacterium tuberculosis.

Figure 1:Analysis flow chart and main results

Male patients were predominant with 63.1% and the average age was 50±15,2 years with extremes of 21 and 86 (Table 2). The over-50 years age group represented 51.6% of patients. Sputum samples represented 95.9% of the treated specimens. HIV status was known for 64 patients of which 15.6% were positive.

Table 2:Demographic and clinical variables of patients with NTM species.

Nested 16S PCR prior to sequencing was performed, for 151 of 155 patients included in study, from 80 clinical specimens and 71 colonies. The test was negative on 33 clinical samples and positive on 118 samples including 47 clinical samples and all 71 cultures (Table 3). Sequencing was performed on sample with clearly positive amplicons, characterizing a specie or a specie group for 95 or 80.5%. For 23 (19.5%), a specific species or specific group of NTM was not able to be characterized (Table 3).

Table 3:NTM Species identified by sequencing.

Characterization of NTM Species by Line Probe Assay GenoType CM/AS

The LPA was performed on 51 strains and species were characterized for 43 (84.3%). The species could not be characterized for 8 patients (15.7%) (Figure 1). MAC was predominant with 25 cases (49%) including 4 species of M. avium and 21 M. intracellulare, respectively 8% and 41% (Figure 2).

Figure 2:NTM Species by LPA GenoType CM/AS.

Comparison Between Sanger Sequencing and LPA

Among the 47 patients who benefited the 2 methods of identification, 32 results had similar patterns. Identification of species with the 2 methods was not possible for 3 patients. Six samples have shown some differences between 2 methods (Table 4), 4 samples (NTM-09-0130, NTM-21-0489, NTM-21-0962, NTM-21-1269), were M. chelonae by sequencing and M. abscessus by LPA CM. One sample (NTM-20-0908) was M. palustre-like by sequencing and M. malmoense by LPA and one (NTM-21-0499) was M. sherrisii by sequencing and M. simiae by LPA. The samples NTM-20-1973 and NTM-22-1804 were identified as M. colombiense by sequencing and M. intracellulare by hybridization (Table 4 & Figure 3).

Figure 3:Alignment of 16SrRNA (rrs) sequence. For NTM-20-1973 (CT2021-01389) and MNT-22-1804 (CT2024-00057) with reference sequences of M. colombiense CECT 3035 and M. intracellulare subsp. intracellulare ATCC 13959.

Table 4:Discordant results between LPA CM/AS and Sanger sequencing.

Note*: NTM=non-tuberculous mycobacteria.

Frequency of Species Identified by the 2 Methods of Identification

MAC was more frequent with 47% (57/122), followed by M. simiae 16 (13.1%), M. fortuitum 9 (7.4%) and M. abscessus 6 (4.9%) (Figure 4). The exact NTM species present could not be identified in 21 patients (17.2%) (Figure 4).

Figure 4:Species grouped by complex or species group.

Discussion

We characterized NTM species detected in our laboratory in patients referred from different health centres in the country. Male sex with 63.1% represented more than half of our patients which is similar to the world tuberculosis report that found 67% of men and 33% of women. (World Health Organization, 2024a). However, female patients were frequently represented with increasing age in several studies [20-22]. The mean age of patients in our study is similar to those reported in other studies [23-26]. The high percentage of clinical samples negative at nested PCR in our study can be explained by a loss of DNA during the multiple stages of sample processing, especially for samples with a low bacillary load. This loss of DNA has been previously reported in some studies [27,28]. The proportion of NTM detected in patients with positive microscopy and MTB not detected by Xpert in our study is slightly lower than that found in other studies [21,29] but higher than that reported in a study in Pakistan [30]. These differences could be explained by the bacillary load of the samples and the techniques used for detection.

Two samples classified as M. species by LPA were identified as M. sherrisii and M. paraense by sequencing. According to the literature, these species cannot be identified by LPA [18,19]. Differential identification of the two closely identic species absessus and chelonae, previously grouped together as M. chelonae-abscessus complex [31] was possible with LPA and not with Sanger sequencing [32,33]. Although this differentiation was possible using hybridization, confusion has been reported with this method [34]. The identification of the 2 species of NTM, M. colombiense by sequencing and M. intracellulare by hybridization in our study, could be explained by nucleotide substitutions in the 16S gene sequence compared with the reference strain ATCC 13950 of M. intracellular subs-intracellulare, and as the target gene for the hybridization test is the 23S gene, the M. colombiense species would not be identifiable. The identification of species such as M. malmoense by the hybridization test and M. palustre by sequencing can be explained by the sharing of the same bands by these 2 species for the GenoType CM kit. In fact, these 2 species are closely identical and are grouped together in the M. simiae complex [35]. In our study, MAC species were more frequent, as in other studies in Mali, Africa and elsewhere [8-16]. Species from M. simiae complex were the most detected after MAC species. Species of these 2 groups are recognized as pathogenic for humans [2].

Conclusion

Species of MAC were the most identified in our study. Molecular methods for TB diagnostic algorithms should also include methods for the detection of cases of NTM. Molecular platforms enabling differential detection of mycobacterial species, could be an opportunity for better management of tuberculosis and mycobacterial diseases.

Acknowledgement

None.

Conflict of Interest

None.

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