CORRESPONDENCE

 

Identifying non-tuberculosis mycobacteria: Is it time to introduce new molecular assays?

 

 

To the Editor: Non-tuberculosis mycobacteria (NTM) are a group of >200 mycobacteria species and subspecies of acid-fast staining bacteria that are not members of the Mycobacterium tuberculosis complex (MTBC) or M. leprae.^[1] Although NTM species remain ubiquitous in the environment, their clinical significance as an emerging opportunistic infection is increasingly recognised, as they may cause pulmonary and extrapulmonary disease.^[2]

The Cape Town National Health Laboratory Service (NHLS) Tuberculosis (TB) laboratory received a sputum specimen collected in error in the community from a 69-year-old woman. On culture, this specimen had an unusual Mycobacterium line probe assay (LPA) banding pattern (Fig. 1). The microscopy (Ziehl Neelsen stain) showed acid-fast bacilli and no contamination. The presence or absence of bands at various sites on the LPA allows for NTMs to be identified and detected. Nevertheless, the banding pattern for this isolate could not be interpreted as the manufacturer did not specify an organism associated with the specific pattern observed.^[3]

To speciate this isolate, it was sent for Sanger sequencing (Central Analytical Facility, Stellenbosch University) of the hsp65 (part of the heat shock protein family) and rpoB (encoding the β-subunit of RNA-polymerase) genes.^[4] The percentage sequence identities to M. septicum from the hsp65 and rpoB genes were 98.88% and 100%, respectively.^[5] In addition, the clinical isolates relationship to similar NTMs was shown in a phylogenetic tree based on the contigs of the rpoB gene (Fig. 2). Regardless of identifying the NTM, the finding remained coincidental, with no treatment or follow-up of the patient, since the NTM was not clinically relevant according to international criteria for pulmonary samples.^[10] This case demonstrates that distinguishing colonisation from NTM disease is challenging,^[10] and to our knowledge the literature is still awaiting a clinically significant M. septicum case from the African continent. A case report of M. septicum and a case series have been published in Asia^[11] and North America,^[12] respectively.

 

 

The assays currently used in South African public health laboratories to identify NTMs are the GenoType Mycobacterium CM and AS LPA (Hain Lifescience, Germany), which are commercial assays based on partially targeting the 23S rRNA sequence.^[4] The LPA technique is well received among laboratories, especially in resource-limited settings, owing to its simplicity in method and interpretation.^¹ However, these DNA hybridisation assays are limited in the spectrum of NTMs they can identify, and are not necessarily tailored for the most prevalent African species. Therefore many NTMs are reported non-specifically as Mycobacterium species without further identification. A systematic review suggests that 29% of pulmonary NTMs from sub-Saharan Africa remained unidentified at the species level.^[131 This is also supported by unpublished data from the NHLS Cape Town TB laboratory, where 14% of 126 NTMs suspected to be clinically relevant in 2020 were documented as non-MTBC Mycobacterium species. Therefore, we conclude that new molecular methods, such as Sanger sequencing and whole genome sequencing,^[41 should be considered to overcome the current limitations in laboratory NTM identification that could impact clinical decision-making and patient outcome.

Acknowledgments. We would like to thank the frontline healthcare workers for their continuous efforts in our communities and for reaching out to this patient.

Author contributions: CJO conceptualised the letter. TD interviewed the patient and requested additional investigations. SS and CJO reviewed and validated laboratory results. WG supervised the sequencing of the genes. All authors edited and approved the final letter.

C J Opperman

S Singh

National Health Laboratory Service, Green Point Laboratory, Cape Town, South Africa; DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa Christoffel.opperman@nhls.ac.za

T Davids

City Health, Community Services and Health, City of Cape Town, South Africa

H Cox

Division of Medical Microbiology, Institute of Infectious Disease and Molecular Medicine; and Wellcome Centre for Infectious Disease Research in Africa, University of Cape Town, South Africa

R Warren

W Goosen

DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa

 

References

1. Tarashi S, Siadat SD, Fateh A. Nontuberculous mycobacterial resistance to antibiotics and disinfectants: Challenges still ahead. Biomed Res Int 2022;2022:8168750. https://doi.org/10.1155/2022/8168750        [ Links ]

2. Ahmed I, Tiberi S, Farooqi J, et al. Non-tuberculous mycobacterial infections - a neglected and emerging problem. Int J Infect Dis 2020;92S:S46-S50. https://doi.org/10.1016/j.ijid.2020.02.022        [ Links ]

3. HAIN Lifescience. GenoType Mycobacterium CM version 2.0, Molecular genetic assay for identification of clinically relevant mycobacterial species from cultured material. Nehren, HAIN Lifescience. https://www.immunodiagnostic.fi/wp-content/uploads/GenoType-CM-V2_kit-insert.pdf (accessed 6 June 2022).         [ Links ]

4. Mwangi ZM, Mukiri NN, Onyambu FG, Wallace BD. Genetic diversity ofnontuberculous mycobacteria among symptomatic tuberculosis negative patients in Kenya. Int J Mycobacteriol 2022;11(1):60-69. https://doi.org/10.4103/ijmy.ijmy_224_21        [ Links ]

5. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215(3):403-410. https://doi.org/10.1016/S0022-2836(05)80360-2        [ Links ]

6. Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 1993;10(3):512-526. https://doi.org/10.1093/oxfordjournals.molbev.a040023        [ Links ]

7. Edgar RC. MUSCLE: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinform 2004;5:113. https://doi.org/10.1186/1471-2105-5-113        [ Links ]

8. Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 2021;38(7):3022-3027. https://doi.org/10.1093/molbev/msab120.         [ Links ]

9. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985;39(4):783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x        [ Links ]

10. Daley CL, Iaccarino JM, Lange C, et al. Treatment ofnontuberculous mycobacterial pulmonary disease: An official ATS/ERS/ESCMID/IDSA clinical practice guideline. Eur Respir J 2020;56(1):2000535. https://doi.org/10.1183/13993003.00535-2020.         [ Links ]

11. Lian L, Deng J, Zhao X, et al. The first case of pulmonary disease caused by Mycobacterium septicum in China. Int J Infect Dis 2013;17(5):e352-4. https://doi.org/10.1016/j.ijid.2012.12.011        [ Links ]

12. Go JR, Wengenack NL, Abu Saleh OM, et al. Mycobacterium septicum: a 6-year clinical experience from a tertiary hospital and reference laboratory. J Clin Microbiol 2020;58(12):e02091-20. https://doi.org/10.1128/JCM.02091-20        [ Links ]

13. Okoi C, Anderson STB, Antonio M, et al. Non-tuberculous Mycobacteria isolated from pulmonary samples in sub-Saharan Africa - a systematic review and meta analyses. Sci Rep 2017;7(1):12002. https://doi.org/10.1038/s41598-017-12175-z        [ Links ]