IJID Regions 3 (2022) 287–292 Contents lists available at ScienceDirect IJID Regions journal homepage: www.elsevier.com/locate/ijregi Prevalence of non-tuberculous mycobacteria among previously treated TB patients in the Gulf of Guinea, Africa B.D. Thumamo Pokama, b , ∗ , D. Yeboah-Manub , b c d S. Ofori , P.W. Guemdjom , P.M. Teyim , L. Lawsone, D. Amiteye f , N.Y. Yhiler a, g , I.C. Djuikoueh, i , A.E. Asuquo j a Department of Medical Laboratory Science, University of Buea, Buea, Cameroon b Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana c Department of Public Health, University of Buea, Buea, Cameroon d Douala Tuberculosis Reference Laboratory, Littoral Region, Cameroon e Zankli Medical Centre, Utako, Abuja, Nigeria f Department of Biomedical Engineering, All Nations University College, Koforidua, Ghana g Department of Allied Health, Biaka University Institute, Buea, Cameroon h Microbiology Department, Faculty of Health Sciences, Université des Montagnes, Bangangte, Cameroon i Foundation Prevention and Control, Cameroon j Department of Medical Laboratory Science, College of Medicine, University of Calabar, Calabar, Nigeria a r t i c l e i n f o a b s t r a c t Keywords: Objective: Differentiation between non-tuberculous mycobacteria ( NTM) and Mycobacterium tuberculosis com- NTM plex (MTBC) is crucial for case management with the appropriate antimycobacterials. This study was undertaken hsp65 sequencing in three West and Central African countries to understand NTM associated with pulmonary tuberculosis in the Drug resistance sub-region. Retreatment patients Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained West/Central Africa from solid and liquid cultures were analysed. The isolates were tested for drug susceptibility, and MTBC were confirmed using IS 6110 . All IS 6110 -negative isolates were identified by 65-kilodalton heat shock protein ( hsp65 ) gene amplification, DNA sequencing and BLAST analysis. Results: Overall, the prevalence of NTM was 16/503 (3.2%), distributed as 2/202 (1%) in Nigeria, 2/158 (1.3%) in Cameroon and 12/143 (8.4%) in Ghana. The main NTM isolates included 5/16 (31.3%) M. fortuitum , 2/16 (12.5%) M. intracellulare and 2/16 (12.5%) M. engbaekii. Eight (57.1%) of the 14 previously treated patients harboured NTM (odds ratio 0.21, 95% confidence interval 0.06–0.77; P = 0.021). Three multi-drug-resistant strains were identified: M. engbaekii, M. fortuitum and M. intracellulare. Conclusion: NTM were mainly found among individuals with unsuccessful treatment. This highlights the need for mycobacterial species differentiation using rapid molecular tools for appropriate case management, as most are resistant to routine first-line antimycobacterials. I d n b c E i N m i ( ( o p a e o 6 h R 2 l ntroduction Non-tuberculous mycobacteria (NTM), recently recognized as a ause of pulmonary and extrapulmonary infection in humans, are mportant emerging pathogens, especially in people living with hu- an immunodeficiency virus/acquired immunodeficiency syndrome HIV/AIDS) ( Lei et al ., 2019 ). The clinical and molecular epidemiol- gy of prevalent pulmonary NTM disease are not as well described s those for pulmonary tuberculosis (PTB) in sub-Saharan Africa ( Okoi t al ., 2017 ) because of diagnostic challenges ( van Ingen, 2013 ). Several∗ Corresponding author. Department of Medical Laboratory Science, Faculty of H 77358743. E-mail address: thumamo@yahoo.fr (B.D.T. Pokam) . ttps://doi.org/10.1016/j.ijregi.2022.05.003 eceived 26 January 2022; Received in revised form 9 May 2022; Accepted 12 May 772-7076/© 2022 Published by Elsevier Ltd on behalf of International Society for I icense ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) ifficulties associated with the use of phenotypic and molecular diag- ostic methods for precise identification of NTM to species level have een linked with shared metabolic and genetic characteristics ( Escobar- scamilla et al. , 2014 ). Moreover, it is difficult to differentiate between TM colonization and NTM disease. Despite these challenges, accurate dentification of the causative species associated with both tuberculosis TB) and NTM is relevant to the proper management and follow-up of atients ( Escobar-Escamilla et al. , 2014 ). In some parts of sub-Saharan Africa where PTB diagnosis is based n clinical symptoms, sputum smear microscopy and (rarely) radiolog-ealth Sciences, University of Buea, P.O. Box 63, Buea, Cameroon. Tel.: + 237 2022 nfectious Diseases. This is an open access article under the CC BY-NC-ND B.D.T. Pokam, D. Yeboah-Manu, S. Ofori et al. IJID Regions 3 (2022) 287–292 i μ p I A t t c I l a d ( i t e i p T u r C a c I ( f o e u ( t r q m p g ( s l t f D r i s T e D N o w U G f g M o c S R t I C a T G g f t 9 ( ( K m i l c w m 9 N 0 D S T M N ( L s 7 w o ( i a L r D i c ( d G s cal findings, NTM may be misdiagnosed and are often treated inap- ropriately as Mycobacterium tuberculosis complex (MTBC) ( Pokam and suquo, 2012 ; Okoi et al ., 2017 ). The clinical and microbiological cri- eria defined by the American Thoracic Society/Infectious Disease So- iety of America to diagnose pulmonary NTM disease are often not fol- owed because of poor laboratory infrastructure. Consequently, anti-TB rugs are administered based on identification of acid-fast bacilli (AFB) Griffith et al. , 2007 ; Pokam and Asuquo, 2012 ). The resulting treatment failure may be misconstrued as drug resis- ance, with the patient placed on an second-line anti-TB drug; this is neffective given the innate resistance of NTM to conventional anti- B drugs ( Griffith et al., 2007 ). Thus, proper identification of NTM is equired for an appropriate treatment protocol to prevent the associ- ted morbidity and mortality that could result from such misdiagnosis. t has been proposed that several treatment regimens may be required or the management of NTM, considering that the genetic make-up of ach strain determines the various drug resistance patterns encountered Griffith, 2010 ). Genotypic methods are available for identification of mycobacte- ia. The 65-kilodalton heat shock protein (hsp65) gene, present in all ycobacteria, has been shown to be useful for the differentiation of enetically-related species ( Ringuet et al ., 1999 ). The unique nucleotide equences of rapidly and slowly growing mycobacteria have been iden- ified at species level ( Plikaytis et al ., 1992 ; Steingrube et al ., 1995 ; evallois et al ., 1997 ), with DNA sequencing analysis providing med- cally relevant sub-specific phylogenetic lineages ( Ringuet et al ., 1999 ). he distribution of distinct NTM species differs geographically ( Okoi t al ., 2017 ), but the cultivation, identification and differentiation of TM from MTBC is not performed routinely in many TB diagnostic lab- ratories in sub-Saharan Africa. Therefore, this study used variability ithin the hsp65 gene to identity NTM species obtained from Cameroon, hana and Nigeria, located in the Gulf of Guinea, Africa. aterials and methods tudy settings and patients The study was carried out between January and December 2017 in hree west and central African countries, namely Ghana, Nigeria and ameroon and included both new and previously treated pulmonary B patients seeking care in various hospitals. Sampling for testing in hana was at the Eastern region of Ghana, specifically the Eastern Re- ional Hospital located in the New Juaben Municipality of Koforidua - he capital of the region. The site in Nigeria was the North Central zone Middle Belt) of the country, including the following states: Benue, Kogi, wara, Nasarawa, Niger, Plateau and Federal Capital Territory. The site n Cameroon was the Littoral region, including Douala, the economic apital. The study was approved by the Scientific and Technical Com- ittee, and the Ethical Committee of the Institutional Review Board of oguchi Memorial Institute for Medical Research (FWA 00001824, IRB 0001276, NMIMR-IRB CPN 007/16-17, IORG 0000908). pecimen culture and drug sensitivity sensitivityA total of 503 isolates (158 from Cameroon, 202 from igeria and 143 from Ghana) were obtained from culture using either owensteinJensen medium (Nigeria and Cameroon) or Middlebrook H9 Broth using BACTEC MGIT 960 (Ghana). Primary identification f MTBC isolates was carried out in Nigeria and Cameroon (but not n Ghana, confirmed by Ziehl-Neelsen staining only) using the SD BIO- INE TB Ag MPT64 RAPID kit (Standard Diagnostics, Inc., Yongin, Ko- ea) based on the manufacturer’s instructions. Drug susceptibility test- ng was carried out for Streptomycin (STR). Isoniazid (INH) Rifampicin RIF) and Ethambutol (ETH) using the proportion method (Nigeria) as escribed previously ( Pokam et al., 2019 ) and BACTEC MGIT 960 in hana using 1.0 μg/ml STR, 0.1 μg/ml INH, 1.0 μg/ml RIF, and 5.0288 g/ml ETH for low drug concentrations and 4.0 μg/ml STR, 0.4 μg/ml NH, and 7.5 μg/ml ETH for high drug concentrations. Rifampicin resis- ance in Cameroon was determined using Xpert MTB/RIF. S6110 PCR assay, hsp65 gene amplification, DNA sequencing, and blast nalysis DNA from each isolate was used for PCR amplification us- ng the insertion sequence 6110 (IS 6110 ) primers described arlier ( Pokam et al ., 2019 ). All IS 6110 -negative strains sus- ected to be NTM were subjected to hsp65 gene amplification sing the Tb11 (5 ′ -CAACGATGGTGTGTCCCAT-3 ′ ) and Tb12 (5 ′ - TTGTCGAACCGCATACCCT-3 ′ ) primers, as well as the polymerase hain reaction (PCR) and cycling condition described previously Otchere et al ., 2017 ). The amplified 441-bp product was confirmed n 1.5% agarose gel electrophoresis, and visualized under short-wave ltraviolet light after ethidium bromide staining. The PCR products of the amplified hsp65 gene were shipped o Macrogen Europe ( https://dna.macrogen-europe.com/eng/ ) for se- uencing. Briefly, the procedure included removal of vector sequences, rocessing into gap files, and editing with gap4 of the Staden package Staden et al ., 2000 ). The consensus hsp65 gene sequence of each iso- ate was saved in FASTA format, and analysed using the National Center or Biotechnology Information nucleotide BLAST against the database of epresentative genomes of microbes, and MegaBLAST selecting highly imilar sequences (Table S1, see online supplementary material). ata analysis The coded data entered into Excel (Microsoft Corp., Redmond, WA, SA) were analysed using SPSS Version 20.0 (IBM, Armonk, NY, USA) or associations between the variables using Chi-squared test for age and ender, and Fisher’s exact test for treatment status and drug resistance f identified NTM. P < 0.05 was considered to indicate statistical signifi- ance with 95% confidence intervals (CI). esults S6110-negative isolates and hsp65 amplification results in the three study reas Following IS 6110 PCR assay of the 503 isolates, 58 (11.5%) were ound to be negative. Of these, 8/158(5.1%) came from Cameroon, /202(4.5%) from Nigeria and 41/143(28.7%) from Ghana. Seven 12.1%) of the 58 IS 6110-negative isolates could not be amplified by the ycobacterium-specific hsp 65 primers, indicating that they do not be- ong to the genus Mycobacterium . Hence, 51 (10.1%) of the 503 isolates ere positive for hsp 65 amplification: 6/158(3.8%) from Cameroon, /202(4.5%) from Nigeria and 36/143(25.2%) from Ghana ( Table 1 ). istribution of MTBC, NTM and Non-Mycobacterial (NM) Species The distribution of the 51 sequenced isolates is shown in Table 2 . hirty-five (68.6%) were MTBC [33 (64.7%) M. tuberculosis and 2 (3.9%) . africanum ], 14 (27.5%) were NTM and 2(3.9%) non mycobacteria one (1.9%) each of Nocardia veterana and Streptomyces sp. ) following equencing and BLASTn. Among the 51 sequenced isolates, 5 (9.8%) ere M. fortuitum strain/subsp. fortuitum , 2 (3.9%) M. intracellulare , 2 3.9%) M. engbaekii , 1 (1.9%) each of M. colombiense, M. gordonae, M. vium, M. paraense, M. peregrinum . istribution and prevalence of hsp65-sequenced isolates in the three ountries The distribution of NTM in each country showed that of the 51 hsp65 - equenced isolates, 2/9 (22.2%) were obtained in Nigeria, 2/6 (33.3%) B.D.T. Pokam, D. Yeboah-Manu, S. Ofori et al. IJID Regions 3 (2022) 287–292 Table 1 Sixty-five kilodalton heat shock protein (hsp65) gene amplification results of insertion sequence 6110 (IS 6110) -negative isolates in three West and Central African countries. No. of isolates Country of origin Isolates under study IS 6110 negative (%) hsp 65 amplification positive (%) Cameroon 158 8 (5.1) 6 (3.8) Nigeria 202 9 (4.5) 9 (4.5) Ghana 143 41 (28.7) 36 (25.2) Total 503 58 (11.5) 51 (10.1) Table 2 N Strain distribution of non-tuberculous mycobacteria (NTM) and non- mycobacteria (NM) species in the Gulf of Guinea. Strains Frequency (%) r s MTBC ( n = 35) T Mycobacterium tuberculosis sensu stricto 33 (64.7) Mycobacterium africanum 2 (3.9) r NTM ( n = 14) e Mycobacterium avium 1 (1.9) Mycobacterium colombiense 1 (1.9) b Mycobacterium engbaekii 2 (3.9) Mycobacterium fortuitum subsp. fortuitum 5 (9.8) w Mycobacterium gordonae 1 (1.9) S Mycobacterium intracellulare strain 2 (3.9) Mycobacterium paraense 1 (1.9) Mycobacterium peregrinum 1 (1.9) D NM ( n = 2) Nocardia veteran 1 (1.9) Streptomyces sp. 1 (1.9) Total 51 a M MTBC, Mycobacterium tuberculosis complex. e c i w o 4 1 t G t t h a u t D t m a o m 2 a t 2 N c 1 w a s t o 6 p t A a m M 1 r t b o i w s t A ( t 1 ( ( m a m N 2n Cameroon and 10/36 (27.8%) in Ghana. Thus, the overall prevalence f NTM was 2/202 (1.0%) in Nigeria, 2/158 (1.3%) in Cameroon and 0/143 (7.0%) in Ghana. Two NM species were also obtained from the hanaian isolates, giving a prevalence of 2/143 (1.4%) ( Table 3 ). Fur- her analysis included the two NM species among the Ghanian NTM, for n overall prevalence of 12/143 (8.4%). emography and association with NTM/NM Among the 51 amplified isolates, 18 (35.3%) and 33 (64.7%) were btained from female and male participants, respectively, aged between 0 and 89 years (mean age 45.14 years). There was no association be- ween gender [9/33 (27.3%) males vs 7/18 (38.9%) females] of the 16 TM/NM compared with MTBC [odds ratio (OR) 0.59, 95% CI 0.17– .99; P = 0.393]. Similarly, the distribution of NTM and MTBC across the ge groups was not significant ( P = 0.259), as shown in Table 4 . However, he age ranges of 35–44 years and 44–54 years were represented with /13 (46.2%) and 5/10 (50%) of the 16 NTM/NM isolates, respectively. ssociation of treatment status and drug resistance with NTM Of the 51 identified isolates, 37 (72.5%) were newly diagnosed and 4 (27.5%) were either previously treated patients or multi-drug resis- ant. Eight of 14 (57.1%) of the 16 patients with NTM/NM were previ- usly treated patients (OR 0.21, 95% CI 0.06–0.77; P = 0.021) compared ith those with MTBC infection. On the other hand, drug resistance be- ween the two groups NTM/NM and MTBC was not associated with STR OR, 1.47, 95%CI, 0.26–8.23, p = 0.999), INH (OR, 0.15 95%CI, 0.02– .49, p = 0.184), RIF (OR, 0.39, 95%CI, 0.08–2.00, p = 0.391) or ETH OR, 0.59, 95%CI, 0.08–4.21, p = 0.999). However, INH [6/16 (37.5%)] nd RIF [4/11 (36.4%)] showed the highest drug resistance among the TM/NM isolates compared to MTBC ( Table 4 ). 289 TM distribution by country, drug resistance and previous treatment status Three NTM were resistant to STR, six were resistant to INH, four were esistant to RIF and two were resistant to ETH. There were three MDR trains: M. engbaekii, M. fortuitum subsp. fortuitum and M. intracellulare. he M. fortuitum subsp. fortuitum MDR strain was found to be equally esistant to STR and ETH, while the M. intracellulare MDR strain was qually resistant to ETH but not STR. Among the patients treated previously, M. perigrimum and M. colom- iense strains were found in Cameroon; M. engbaekii and M. intracellulare ere found in Nigeria; and M. fortuitum, M. paraense, M. engbaekii and treptomyces sp. were found in Ghana ( Figure 1 ). iscussion NTM are an important cause of pulmonary diseases worldwide, and re being isolated increasingly. They are often mistakenly treated as TBC in countries devoid of laboratory competence for species differ- ntiation ( Pokam and Asuquo, 2012 ). This study showed an overall prevalence of NTM of 3.2% in three ountries in the Central and West African region. The prevalence of NTM as lowest in Nigeria at 1%; previous research reported prevalence of .1% in a South-South state ( Pokam and Asuquo, 2012 ), and 15% across he country ( Aliyu et al ., 2013 ). As the present study included a collec- ion of strains cultured previously from PTB patients, some isolates may ave been excluded following primary identification in the laboratory sing MTB protein 64 (mpt64) antigen, differentiating NTM from MTBC hrough immunochromatographic methods. Nevertheless, 1% escaped his prior screening, probably as a result of the polymorphisms of the pt64 gene in MTBC which lead to changes in the antigen produced and lterations in related functions ( Jiang et al ., 2013 ). Mutation within the pt64 gene has been shown to lead to incomplete protein production as result of deletion of the C-terminal region of the protein ( Hirano et al ., 004 ). The similar prevalence of NTM of 1.3% observed in Cameroon an be explained by the fact that isolates in both Cameroon and Nigeria ere cultured using Lowenstein–Jensen medium with prior exclusion of ome NTM (as rapid growers), whereas in Ghana, with NTM prevalence f 8.4%, BACTEC MGIT liquid was used and no isolates were excluded rior to sequencing. Information about NTM epidemiology in Cameroon is scarce, al- hough a study reported these organisms in cattle from four abattoirs cross the country and hypothetically linked their transmission to hu- ans ( Egbe et al ., 2016 ). Egbe et al. reported M. fortuitum, M. gordonae, . mucogenicum, M. phlei and M. scrofulaceum , whereas the present study eported M. colombiense and M. peregrinum . Although the area studied y Egbe et al. was different from that in the present study, the high nteraction between animals, the environment and humans has been hown to pose a risk of NTM transmission from animals to humans in frica ( Katale et al ., 2014 ). M. colombiense, a new species belonging to he M. avium complex (MAC), has been isolated in children in France Vuorenmaa et al ., 2009 ) and Spain ( Esparcia et al ., 2008 ). Underesti- ation of emerging MAC species has been highlighted due to the lack of odern molecular techniques for their identification ( Vuorenmaa et al ., 009 ). B.D.T. Pokam, D. Yeboah-Manu, S. Ofori et al. IJID Regions 3 (2022) 287–292 Table 3 Distribution and prevalence of non-tuberculous mycobacteria (NTM) and non-mycobacteria (NM) in each studied country. Isolates/country Nigeria Cameroon Ghana Total MTBC M. tuberculosis ( n = 6) M. tuberculosis ( n = 4) M. tuberculosis ( n = 23) 35 M. africanum ( n = 1) M. africanum ( n = 1) NTM M. engbaekii ( n = 1) M. colombiense ( n = 1) M. avium ( n = 1) 14 M. intracellulare ( n = 1) M. peregrinum ( n = 1) M. engbaekii ( n = 1) M. fortuitum ( n = 5) M. gordonae ( n = 1) M. intracellulare ( n = 1) M. paraense ( n = 1) NM Nocardia veterana ( a n = 1) 2 Streptomyces a sp. ( n = 1) Total 9 6 36 51 Overall prevalence 2/202 (1%) 2/158 (1.3%) 12/143 (8.4%) 16/503 (3.2%) (NTM/NM) MTBC, Mycobacterium tuberculosis complex. a Both included in the prevalence calculation of NTM and further analysis. Table 4 Association of age, gender, treatment status and drug resistance with non-tuberculous mycobacteria (NTM) in the three countries. Variables NTM/NM (%) MTBC (%) Total Odds ratio (95% CI) P -value Gender 0.393 Male 9 (27.3) 24 (72.7) 33 0.59 (0.17–1.99) Female 7 (38.9) 11 (61.1) 18 Total 16 35 51 Age (years) 0.259 < 25 1 (33.3) 2 (66.7) 3 1.1 (0.09–13.09) 0.686 25–34 1 (8.3) 11 (91.7) 12 0.15 (0.02–1.24) 0.075 35–44 6 (46.2) 7 (53.8) 13 2.4 (0.65–8.88) 0.298 45–54 5 (50) 5 (50) 10 2.73 (0.66–11.27) 0.253 55–64 2 (28.6) 5 (71.4) 7 0.86 (0.15–4.97) 0.618 > 64 1 (16.7) 5 (83.3) 6 0.4 (0.04–3.74) 0.651 Total 16 35 51 Treatment status 0.021 ND 8 (21.6) 29 (78.4) 37 0.21 (0.06–0.77) PT/TMDR 8 (57.1) 6 (42.9) 14 Total 16 35 51 Drug resistance STR 0.999 S 4 (28.6) 10 (71.4) 14 1.47 (0.26–8.23) R 3 (21.4) 11 (78.6) 14 Total 7 21 28 INH 0.184 S 1 (8.3) 11 (91.7) 12 0.15 (0.02–1.49) R 6 (37.5) 10 (62.5) 16 Total 7 21 28 RIF 0.391 S 4 (18.2) 18 (81.8) 22 0.39 (0.08–2.00) R 4 (36.4) 7 (63.6) 11 Total a 8 25 33 ETH 0. 999 S 5 (22.7) 17 (77.2) 22 0.59 (0.08–4.21) R 2 (33.3) 4 (66.7) 6 Total 7 21 28 NM, non-mycobacteria; MTBC, Mycobacterium tuberculosis complex; ND, newly diagnosed; PT/TMDR, previously treated or treated as multi-drug-resistant strains; CI, confidence interval; STR, streptomycin; INH, isoniazid; RIF, rifampicin; ETH, ethambutol; S, sensitive; R, resistant. a Rifampicin sensitivity alone, carried out using GeneXpert in Cameroon. c e t 1 t f w o s I r M e i i M. peregrinum has been isolated from sputum samples in the East- rn region of China ( Shao et al ., 2015 ). It reportedly represents up to 9% of isolates from human respiratory sites in the USA, despite the act that its clinical significance is unknown, probably due to the lack f large evaluation studies ( Wallace et al ., 2005 ). M. peregrinum type isolates have shown approximately 97.1% identity with M. fortuitum. . fortuitum, a rapidly growing mycobacteria, was the most commonly solated (9.8%) NTM in this study, and all isolates were found in Ghana,290 ontrary to a previous study in Ghana that did not isolate any M. for- uitum ( Otchere et al ., 2017 ). Instead, Otchere et al . found that M. in- racellulare (30.2%) was the most prevalent NTM in Ghana, whereas it as much less prevalent in the present study. This is surprising con- idering the ubiquitous nature of M. fortuitum, which has been widely ecovered from humans as well as animals ( da Costa et al ., 2013 ; Katale t al ., 2014 ). Other NM species ( Streptomyces sp. and N. veterana ) were solated in Ghana, and further evaluation is required to determine their B.D.T. Pokam, D. Yeboah-Manu, S. Ofori et al. IJID Regions 3 (2022) 287–292 Figure 1. Distribution of non-tuberculous mycobacteria/non-mycobacteria isolates by country, drug resistance and previous treatment status. STR, streptomycin; INH, isoniazid; RIF, rifampicin; ETH, ethambutol; MDR, multi-drug resistant. i G l s n d ( s t a p M p b b o A s s M N s p ( a c 2 w s t i r a c w u s a s ( s m mportance in TB-like infection. An earlier study showed that some iso- ates from sputum-smear-positive patients considered to be MTB were ot members of the genus Mycobacterium , but resembled Nocardia spp. Pokam and Asuquo, 2012 ). Thus, there is a need for newer molecular echniques to unfold these hidden and unrecognized infections in TB atients, especially in sub-saharan Africa. In contrast to Ghana and Cameroon with limited data on the distri- ution of NTM, several studies in Nigeria have reported the prevalence f NTM in different parts of the country ( Pokam and Asuquo, 2012 ; liyu et al ., 2013 ; Cadmus et al ., 2016 ). Aliyu et al . (2013) and other tudies have associated the Harmattan season with the occurrence of TM cases. M. intracellulare reported in this study has been described reviously, and the absence of M. abscessus and M. fortuitum, which re commonly found in Nigeria ( Pokam and Asuquo, 2012 ; Aliyu et al ., 013 ), may be due to the low prevalence reported previously as a re- ult of prior elimination of NTM in the present study. In this study, the dentification of a new species – M. engbaekii – highlights the diversity nd large spectrum of NTM that can be encountered in Nigeria. Drug resistance was observed among NTM isolated in this study, with p to 85.7% resistance to INH, 42.9% resistance to STR and 50% re- istance to RIF. It has been shown that most NTM do not respond to tandard anti-TB drugs ( Viveiros et al ., 2003 ). In support of the present tudy, Otchere et al . (2017) reported high resistance to INH and RIF in291 hana, suggesting the need for proper species identification and drug ensitivity evaluation in patients. MAC infections are difficult to treat ue to the intrinsic multi-drug resistance of the organism. The present tudy showed that virtually all the species were resistant to one or more nti-TB drugs, and M. engbaekii, M. fortiutum and M. intracellulare were DR. Resistance of M. intracellulare to INH and RIF has been reported reviously in Ghana ( Otchere et al ., 2017 ), highlighting that NTM may e MDR ( Shahraki et al ., 2015 ). This study also showed that NTM (57.1%) were significantly as- ociated with previous treatment or MDR-TB. With the exception of . avium, M. gordonae, M. paraense and N. veterana in Ghana, all the pecies isolated in each country were MDR. In Nigeria, Cadmus et al. 2016) noted, in line with the present study, that 40% of NTM were re- overed from patients diagnosed as new TB cases, and 33% of patients ere diagnosed as relapsed TB, buttressing the public health implica- ions of TB misdiagnosis in NTM-infected patients. This appears to be ecurrent in the country, as Pokam and Asuquo (2012) also noted the onsequences of directly observed treatment in AFB-positive individuals ithout further proof of the organisms involved in TB-like symptoms. Approximately 69% of patients infected with NTM in this study were ged between 35 and 54 years, with more males (56%) than females 44%). This is contrary to a study undertaken in Brazil, which found that ore females harboured NTM compared with males (72.4% vs 37.4%). B.D.T. Pokam, D. Yeboah-Manu, S. Ofori et al. IJID Regions 3 (2022) 287–292 T S w d t t n b R c A t m C S d H D a c c E M E d C E G t w G t H m e n J p K i p o L n o O d O C P A P P w N R i S F S t D S i S r p v A V V L P W m t he study found that the average age of NTM patients was 52 years, hich was significantly higher than that of TB patients (39 years). The ifference can be explained by the younger age of the African popula- ion, which may be connected to the implications of poverty and mal- utrition among younger age groups, and the consequent effect on the urden of infectious diseases such as NTM, as in the present study. In ontrast, in developed countries, elderly individuals respond to infec- ions less favourably than young individuals, mainly as a result of im- unosenescence ( da Costa et al ., 2013 ). tudy limitations The results of this study should be interpreted with caution as the IV status of the patients was not included (HIV results were only avail- ble for Nigeria, and hence too few individuals were identified for in- lusion in this analysis). Nevertheless, of the nine Nigerian patients in- luded in this study, two were HIV positive, one of whom harboured . intracellulare. Repeated isolation of NTM is required for definitive iagnosis of the isolate in a patient. onclusion This study highlights the importance of hsp65 sequencing in the iden- ification of NTM in the Gulf of Guinea to prevent the burden associated ith management of presumed relapse or MDR patients. This may help o prevent the far-reaching consequences for patients and the develop- ent of drug resistance in this sub-region of Africa, where the means to ffectively combat the emergence and threat of MDR across the conti- ent is limited. A full evaluation of the NTM species circulating in this art of the world is needed urgently. The need for rapid and correct dentification of the causative Mycobacterium spp. cannot be overem- hasized considering that each infection is unique in terms of choice f drugs and duration of therapy. To achieve this, laboratory capacity eeds to be increased, especially in the Gulf of Guinea where persistence f this gap will continue to lead to morbidity and mortality of overbur- ened patients that could have been avoided. onflict of interest statement None declared. cknowledgements This work is dedicated to Prof. Lovett Lawson (one of the authors), ho died before submission of this paper, who provided isolates from igeria used in this study and contributed tremendously to TB research n his country. May his soul rest in peace. unding This work was supported by the Bill and Melinda Gates Foundation o BDTP under the Postdoctoral and Postgraduate Training in Infectious iseases Research awarded to the Noguchi Memorial Institute for Med- cal Research (Global Health Grant No. OPP52155). The funder had no ole in study design, data collection and analysis, decision to publish or reparation of the manuscript. uthor contributions BDTP, DYM and AEA conceived and designed the experiment. 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