Prevalence estimation of Tropheryma whipplei in duodenal biopsy tissues of Koreans.

Whipple's disease caused by Tropheryma whipplei is difficult to diagnose because of a broad spectrum of manifestations and non-specific clinical signs. In the current global era, the incidence of duodenal infection/inflammation caused by T. whipplei in Korea may has been underestimated. Here we estimated the prevalence of T. whipplei in duodenal biopsy tissues of Koreans using real-time PCRs (RT-PCRs). A total of 252 duodenal biopsy tissues were collected from Korean patients who underwent esophagogastroduodenoscopy and duodenal biopsy. DNA extracted from the duodenal biopsy tissues was analyzed using three RT-PCRs targeting T. whipplei-specific regions of the 16S-23S rRNA intergenic spacer, hsp65, and Dig15 in parallel. In the samples positive in RT-PCRs, direct sequencing was performed for each RT-PCR target. The prevalence of T. whipplei was estimated based on the RT-PCR and sequencing results. Among the analyzed samples, T. whipplei was not detected. The prevalence of T. whipplei in duodenal biopsy tissues of Koreans was estimated to be less than 0.4%. This is the first study to attempt to detect T. whipplei in duodenal biopsy tissues of Koreans and estimate its prevalence. Our findings infer that while T. whipplei carriers exist in Korea, the incidence of duodenal infection/inflammation caused by T. whipplei is extremely rare.

Hsp65-producing Lactococcus lactis inhibits experimental autoimmune encephalomyelitis by preventing cell migration into spinal cord.

Multiple sclerosis is an autoimmune disease that affects the central nervous system. Because of its complexity and the difficulty to treat, searching for immunoregulatory responses that reduce the clinical signs of disease by non-aggressive mechanisms and without adverse effects is a scientific challenge. Herein we propose a protocol of oral tolerance induction that prevented and controlled MOG-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. The genetically modified strain HSP65-producing Lactococcus lactis was orally administered for 5 consecutive days either before or during disease development in mice. Both protocols of feeding HSP65 resulted in significant reduction in the clinical score of EAE. Frequencies of LAP+CD4+Foxp3- regulatory T cells were higher in spleens and inguinal lymph nodes of fed mice. In addition, intravital microscopy showed that adherence of leukocytes to venules in the spinal cord was reduced in orally treated mice. Oral treatment with HSP65-producing L.lactis prevented leukocytes to leave the secondary lymphoid organs, therefore they could not reach the central nervous system. Despite the inhibition of pathological immune response that drive EAE development, activated T cells were at normal frequencies suggesting that oral tolerance did not induce general immunosuppression, but it led to specific control of pathogenic T cells. Our results indicate a novel therapeutic strategy to prevent and control autoimmune diseases such as multiple sclerosis.

Prevalence and detection of Tropheryma whipplei in the stools of Korean patients with diarrhea using real-time PCRs.

BACKGROUND: The prevalence of Tropheryma whipplei varies depending on age, region, and underlying disease. We estimated the prevalence of T. whipplei in the stools of Korean patients with diarrhea using real-time PCR (RT-PCR) and compared three RT-PCR targets, rpoB, hsp65, and Dig15. METHODS: A total of 1404 nucleic acid samples extracted from the stools of Korean patients with diarrhea were tested using an initial RT-PCR targeting T. whipplei-specific regions of 16S-23S rRNA intergenic spacer. Subsequently, the samples positive for the initial RT-PCR were tested using the follow-up RT-PCRs targeting rpoB, hsp65, and Dig15 and analyzed by sequencing to confirm the presence of T. whipplei. We estimated the prevalence of T. whipplei and compared them according to gender and age. We also compared the performance of three targets in the follow-up RT-PCRs. RESULTS: T. whipplei was detected in 1.4% of all samples (20 of 1404), and there were no differences according to gender and age. In pediatric samples (≤ 19 years), T. whipplei was detected higher in children aged 6-19 than in those aged 1-5 (2.7% vs. 0.7%, P = 0.01). Sensitivities of the rpoB, hsp65, and Dig15 RT-PCR were 50.0%, 85.0%, and 95.0%, respectively; specificities were 100.0%, 100.0%, and 84.6%, respectively. CONCLUSIONS: This is the first study that estimated the prevalence of T. whipplei in the stools of Korean patients with diarrhea. This study demonstrated the presence of T. whipplei in stools of Koreans, even though the bacterium was detected low. The RT-PCRs targeting hsp65 and Dig15 showed reliable performance, and a multiplex PCR including these targets is expected to be useful for T. whipplei detection.

Molecular identification of Mycobacterium spp. isolated from Brazilian wild boars.

Wild boars (Sus scrofa) are susceptible to mycobacterial infections, including tuberculous and non-tuberculous mycobacteria. Recently, Mycobacterium spp. infections were described in Brazilian wild boars, which can act as bacterial reservoirs. Here, we aim to characterize 15 Mycobacterium spp. isolates from Brazilian wild boars' tissues through partial sequencing of the heat shock protein 65 (hsp65) gene and phylogenetic analysis. The isolates were classified as M. tuberculosis (33.3%), M. colombiense (33.3%), M. avium subsp. hominissuis (13.3%), M. parmense (13.3%) and M. mantenii (6.66%). The isolates classified as M. tuberculosis were confirmed as variant bovis by PCR. At phylogenetic analysis some isolates formed separated clades, indicating genetic variability. Different Mycobacterium species were recovered from wild boars circulating in Brazil, including mycobacteria associated to zoonotic infections, such as M. tuberculosis. In addition, this is the first report in Brazilian wild boars on M. mantenii and M. parmense detection, two recently described pathogenic mycobacteria. However, the isolates' genetic diversity-i.e. identities lower than 100% when compared to reference sequences-suggests that other genotyping tools would allow a deeper characterization. Nonetheless, the reported data contributes to the knowledge on mycobacterial infections in wild boars from Brazil.

First case report of prosthetic valve endocarditis caused by Mycobacterium wolinskyi.

To date, only 26 cases of Mycobacterium wolinskyi infections have been reported in humans. We herein report a first case of prosthetic valve endocarditis due to this organism after cardiovascular surgery. An 82-year-old man presented with repeat episodes of syncope and fever after aortic valve replacement, mitral valve replacement, left atrial appendage closure, and pulmonary vein isolation. Blood cultures maintained in aerobic bottles were repeatedly positive after 90-100 hours, and Gallium scan revealed abnormal accumulations in the sternum and left testis. While colonies formed by culturing the fluid of the parasternal area and blood cultures revealed gram-positive rods, we could not analyze the colony using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). M. wolinskyi was finally identified on 16S rRNA, hsp65, and rpoB gene sequencing. We treated the patient with multiple antimycobacterial drugs, i.e., amikacin, imipenem, and clarithromycin for 6 weeks, which was changed to oral ciprofloxacin and minocycline for 12 months. This case highlights the need to consider rapidly growing mycobacteria, including M. wolinskyi, if chronic fever persists from weeks to months after surgery, the blood culture is positive, and the organism is not identified. In addition, sequencing the 16S rRNA, hsp65, and rpoB genes is essential for diagnosis.

Invasive Lactococcus lactis producing mycobacterial Hsp65 ameliorates intestinal inflammation in acute TNBS-induced colitis in mice by increasing the levels of the cytokine IL-10 and secretory IgA.

AIMS: To investigate the anti-inflammatory activity of an invasive and Hp65-producing strain Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) in acute 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis in mice as an innovative therapeutic strategy against Crohn's disease (CD). METHODS AND RESULTS: The pXYCYT:Hsp65 plasmid was transformed into the L. lactis NCDO2118 FnBPA+ strain, resulting in the L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain. Then, the functionality of the strain was evaluated in vitro for Hsp65 production by Western blotting and for invasion into Caco-2 cells. The results demonstrated that the strain was able to produce Hsp65 and efficiently invade eukaryotic cells. Subsequently, in vivo, the anti-inflammatory capacity of the recombinant strain was evaluated in colitis induced with TNBS in BALB/c mice. Oral administration of the recombinant strain was able to attenuated the severity of colitis by mainly reducing IL-12 and IL-17 levels and increasing IL-10 and secretory immunoglobulin A levels. CONCLUSIONS: The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to a reduction in inflammatory damage in experimental CD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, which used L. lactis for the production and delivery of Hsp65, has scientific relevance because it shows the efficacy of this new strategy based on therapeutic protein delivery into mammalian enterocytes.

Isolated mediastinal lymphadenitis caused by Mycobacterium malmoense in an immunocompromised child.

Mycobacterium malmoense is a nontuberculous mycobacteria (NTM), that is uncommon in areas other than Northern Europe. We describe the case of mediastinal lymphadenitis caused by M. malmoense in a 4-year-old boy who has a past medical history of disseminated Bacille de Calmette et Guérin (BCG) infection. He presented with persistent high fever and computed tomography revealed mediastinal lymphadenopathy. We identified M. malmoense by hsp65 gene analysis from a lymph node biopsy sample. We treated him with rifampicin, ethambutol and clarithromycin with reference to the guidelines of the British Thoracic Society. M. malmoense can cause severe infections including mediastinal lymphadenitis in children with susceptibility to acid-fast bacteria (AFB).

Identification of mycobacteria species by molecular methods.

In this study, mycobacteria, which were previously identified as Mycobacterium tuberculosis complex (MTC), and mycobacteria other than tuberculosis (MOTT) with cord factor and the p-nitro-alpha-acetyl-amino-beta-hydroxypropiophenone (NAP) test were reanalysed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis method in order to confirm the identification, and at the same time, species accepted as MOTT were identified. Although the results of the NAP test were obtained within 3-5 days, the PCR-RFLP results were obtained in 1 day. Ten species identified as MTC with the NAP test and cord factor were confirmed with the PCR-RFLP method. Fourteen species accepted as MOTT were identified as Mycobacterium species with the evaluation of the bands observed after the restriction of PCR product with the PCR-RFLP method. These were as follows: three species Mycobacterium intracellulare type I, two species Mycobacterium phlei, two species Mycobacterium kansasii, one species Mycobacterium fortuitum type I, one species Mycobacterium gordonae type I, one species Mycobacterium abscessus type I, one species Mycobacterium scrofulaceum, one species Mycobacterium szulgai type I, one species Mycobacterium avium type II, and one species Mycobacterium terrae. Hence, the results of both the cord factor and the NAP test were confirmed with the molecular method, and at the same time, mycobacteria species identification was made by determining the fastest, easiest, and the most accurate result-giving method. Because PCR-RFLP is a very rapid method that provides exact identification of mycobacteria species, it can be performed in routine procedures.

Identification of nontuberculous mycobacteria using multilocous sequence analysis of 16S rRNA, hsp65, and rpoB.

BACKGROUND: The isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased, and they now are considered significant opportunistic pathogens. The aims of this study were to develop a database and interpretive criteria for identifying individual species. In addition, using clinical isolates, we evaluated the clinical usefulness of 16S rRNA, hsp65, and rpoB as target genes for this method. METHODS: The sequences of NTM for 16S rRNA, hsp65, and rpoB were collected from GenBank and checked by manual inspection. Clinical isolates collected between 2005 and 2010 were used for DNA extraction, polymerase chain reaction, and sequencing of these three genes. We constructed a database for the genes and evaluated the clinical utility of multilocus sequence analysis (MLSA) using 109 clinical isolates. RESULTS: A total 131, 130, and 122 sequences were collected from GenBank for 16S rRNA, hsp65, and rpoB, respectively. The percent similarities of the three genes ranged from 96.57% to 100% for the 16S rRNA gene, 89.27% to 100% for hsp65, and 92.71% to 100% for rpoB. When we compared the sequences of 109 clinical strains with those of the database, the rates of species-level identification were 71.3%, 86.79%, and 81.55% with 16S rRNA, hsp65, and rpoB, respectively. We could identify 97.25% of the isolates to the species level when we used MLSA. CONCLUSION: There were significant differences among the utilities of the three genes for species identification. The MLSA technique would be helpful for identification of NTM.

Evaluation of MALDI-TOF MS for identification of nontuberculous mycobacteria isolated from clinical specimens in mycobacteria growth indicator tube medium.

Nowadays, there is a rising worldwide incidence of diseases caused by nontuberculous mycobacteria (NTM) species, especially in immunocompromised patients and those with underlying chronic pulmonary diseases. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) became a method of choice for the identification of NTM species. The aim of this study was to evaluate MALDI-TOF MS for the identification of NTM isolates compared to the PCR-restriction enzyme analysis (PRA)-hsp65 method. In this study, a total of 152 NTM strains isolated from various clinical specimens were retrospectively analysed. MALDI-TOF MS successfully identified 148 (97.4%) of the 152 NTM isolates but failed to identify four (2.6%) of them. Bruker mycobacteria library gave spectral scores higher than 2.0 for 45 (29.6%) of NTM isolates, between 1.6 and 2.0 for 98 (64.5%) of NTM isolates, and lower than 1.6 for nine (5.9%) NTM isolates. The discordant results between MALDI-TOF MS and PRA-hsp65 analysis were confirmed by sequence analysis. In conclusion, MALDI-TOF MS is a technique capable of performing accurate, rapid, cost-effective, and easy identification of NTM isolates.

Comparison of restriction enzyme pattern analysis and full gene sequencing of 16S rRNA gene for Nocardia species identification, the first report of Nocardia transvalensis isolated of sputum from Iran, and review of the literature.

Nocardial infections occur in different organs of the body and are common in immune disorder diseases of individuals. The aim of this study was to assess Nocardia species identification by phenotypic tests and molecular techniques applied to nocardiosis in Iranian patients. In the current study, various clinical samples were collected and cultured on conventional media and using the paraffin baiting method. Various phenotypic tests were performed. For accurate identification at the species level, restriction fragment length polymorphisms (RFLP) in the hsp65 and partial 16S rRNA genes and full gene sequencing of the 16S rRNA gene were used. Twenty-seven Nocardia spp. were isolated and analysis of phenotypic tests results showed Nocardia asteroides complex, Nocardia otitidiscaviarum, Nocardia nova, and Nocardia spp. New RFLP patterns of Nocardia strains with hsp65 and partial 16S rRNA genes were obtained. Full gene sequencing of the 16S rRNA gene identified Nocardia cyriacigeorgica, N. otitidiscaviarum, Nocardia farcinica, Nocardia transvalensis, and N. nova. Nocardia infections are rarely reported and this genus is the cause of various illnesses. Accurate identification of Nocardia spp. is important for epidemiology studies and treatment. It should also be noted that some species may have similar RFLP patterns; therefore, full gene sequencing of the 16S rRNA gene is necessary for confirmation.

First report of isolation of Mycobacterium canariasense from hospital water supplies.

Mycobacterium canariasense was first isolated as a novel species in 2004 from clinical specimens in Spain. Since then there have only been a few additional reports from Spain, the USA, and Lebanon on the isolation of this rare species from clinical specimens. We herein present the first report on isolation of this organism from hospital water, which provides evidence for determining the natural habitat of this rare species. The water samples were collected from hospital departments and cultured on Löwenstein-Jensen and Sauton's media. The isolates, i.e. WP5, WP20, and AW2-3, were subjected to identification by conventional and molecular tests including sequencing analysis of 16S rRNA. The water isolates revealed phenotypic and molecular features consistent with M. canariasense including a genus-specific amplicon of the hsp65 gene and 100% similarities with those of M. canariasense CIP: 107998(T) 16S rRNA gene sequences. The current report might be of value in tracing the probable source of infection in patients.

Isolation and identification of nontuberculous mycobacteria from raw milk and traditional cheese based on the 16S rRNA and hsp65 genes, Tehran, Iran.

As an important source of human food, milk can be a carrier of human pathogenic bacteria, including tuberculous and nontuberculous mycobacteria (NTM), in its raw and unpasteurized state. In this research, 175 raw milk samples and 175 traditional cheese samples were collected from traditional dairy stores in 22 regions of Tehran in a 9- month period from August 2019 to May 2020. Samples were prepared and transferred to a specialized laboratory, where they were inoculated in Lowenstein-Jensen (LJ) medium containing glycerol or sodium pyruvate, as well as Herrold's egg-yolk with and without Mycobactin J. to determine the sample's identity of samples. The recommended 16S rRNA (1436 bp) and hsp65 (644 bp) gene fragments from the positive isolates identified in Ziehl-Neelsen (Z-N) staining were amplified and sequenced using PCR and compared with the sequences of the gene fragments of reference strains available in the global GenBank database. No mycobacterial species were isolated from traditional cheese samples in microbial culture. In case of raw milk samples, a total of four bacteria were collected, all of which were found in the genetic differential testing to be NTM, including n = 1 Mycobacterium heraklionense, n = 2 Mycolicibacterium fortuitum, and n = 1 Mycobacterium thermoresistibile. The analysis of the results obtained by isolate sequencing using the 16S rRNA gene showed higher discriminatory power and percentage similarities in the identification of the isolates than the hsp65 gene.

Detection of Mycobacterium bovis in nasal swabs from communal goats (Capra hircus) in rural KwaZulu-Natal, South Africa.

Animal tuberculosis, caused by Mycobacterium bovis, presents a significant threat to both livestock industries and public health. Mycobacterium bovis tests rely on detecting antigen specific immune responses, which can be influenced by exposure to non-tuberculous mycobacteria, test technique, and duration and severity of infection. Despite advancements in direct M. bovis detection, mycobacterial culture remains the primary diagnostic standard. Recent efforts have explored culture-independent PCR-based methods for identifying mycobacterial DNA in respiratory samples. This study aimed to detect M. bovis in nasal swabs from goats (Capra hircus) cohabiting with M. bovis-infected cattle in KwaZulu-Natal, South Africa. Nasal swabs were collected from 137 communal goats exposed to M. bovis-positive cattle and 20 goats from a commercial dairy herd without M. bovis history. Swabs were divided into three aliquots for analysis. The first underwent GeneXpert® MTB/RIF Ultra assay (Ultra) screening. DNA from the second underwent mycobacterial genus-specific PCR and Sanger sequencing, while the third underwent mycobacterial culture followed by PCR and sequencing. Deep sequencing identified M. bovis DNA in selected Ultra-positive swabs, confirmed by region-of-difference (RD) PCR. Despite no other evidence of M. bovis infection, viable M. bovis was cultured from three communal goat swabs, confirmed by PCR and sequencing. Deep sequencing of DNA directly from swabs identified M. bovis in the same culture-positive swabs and eight additional communal goats. No M. bovis was found in commercial dairy goats, but various NTM species were detected. This highlights the risk of M. bovis exposure or infection in goats sharing pastures with infected cattle. Rapid Ultra screening shows promise for selecting goats for further M. bovis testing. These techniques may enhance M. bovis detection in paucibacillary samples and serve as valuable research tools.

Bacille Calmette-Guérin/DNAhsp65 prime-boost is protective against diabetes in non-obese diabetic mice but not in the streptozotocin model of type 1 diabetes.

Type I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime-boost strategy involving bacille Calmette-Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non-obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non-immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime-boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases.

Identification of Significant Pathogenic Nontuberculous Mycobacteria Species from Presumptive TB Patients Using Partial hsp65 Gene Sequencing.

Purpose: To date, the diagnosis of nontuberculous mycobacteria (NTM) disease primarily relies on clinical symptoms and radiological features. Our objective was to apply a sequence-based analysis method by using partial gene sequencing of heat shock protein 65 (hsp65) to identify NTM species. Patients and Methods: A total of 32 stored isolates obtained from individuals suspected of having pulmonary NTM infection were subjected to solid Ogawa culture. Genomic DNA from each sample was extracted and used in a conventional polymerase chain reaction (PCR) targeting a specific region of hsp65 gene. Identified amplicons from the PCR were then subjected to targeted sequencing. Analysis of the obtained hsp65 sequence was performed using DNA Baser tool. The consensus sequences obtained were compared to references in the GenBank NCBI database to determine NTM species. Results: We identified several important NTM species which posses opportunistic characteristics. M. abscessus and M. chelonae are the most frequent NTM species identified in this study (40.63% and 18.75%, respectively). These two species have the potential to cause significant infections in human, ranging from opportunistic pulmonary infection to localized skin infection. Additionally, pathogenic NTM members of M. fortuitum group (MFG), M. avium, M. intracellulare, M. kansasii, and M. celatum were also found among all identified species. Conclusion: Sequence-based analysis is a promising method for identifying species of NTM. The hsp65 gene has a high discriminatory power to identify opportunistic pathogen NTM species in specimens in Indonesia. Consequently, hsp65 partial gene sequencing is considerable as an alternative and reliable approach for NTM speciation.

Accurate subspecies-level identification of clinically significant Mycobacterium avium and Mycobacterium intracellulare by whole-genome sequencing.

Whole genome sequencing (WGS) of Mycobacterium avium complex (MAC) isolates in the clinical laboratory setting allows for rapid and reliable subspecies identification of a closely related complex of human pathogens. We developed a bioinformatics pipeline for accurate subspecies identification and tested 74 clinical MAC isolates from various anatomical sites. We demonstrate that reliable subspecies level identification of these common and clinically significant MAC isolates, including M. avium subsp. hominissuis (most dominant in causing lower respiratory tract infections in our cohort), M. avium subsp. avium, M. intracellulare subsp. intracellulare, and M. intracellulare subsp. chimaera, can be achieved by analysis of only two marker genes (rpoB and groEL/hsp65). We then explored the relationship between these subspecies and anatomical site of infection. Further, we conducted an in silico analysis and showed our algorithm also performed well for M. avium subsp. paratuberculosis but failed to consistently identify M. avium subsp. silvaticum and M. intracellulare subsp. yongonense, likely due to a lack of available reference genome sequences; all the 3 subspecies were not found in our clinical isolates and rarely reported to cause human infections. Accurate MAC subspecies identification may provide the tool and opportunity for better understanding of the disease-subspecies dynamics in MAC infections.

Safety and Immunogenicity of an In Vivo Muscle Electroporation Delivery System for DNA-hsp65 Tuberculosis Vaccine in Cynomolgus Monkeys.

A Bacille Calmette-Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime-boost regimen, to help the induction of a stronger cellular immune response.

Molecular identification and clinical significance of Mycobacterium seoulense strains from patients with nontuberculous mycobacterium infections.

PURPOSE: To investigate the clinical significance of Mycobacterium seoulense (M. seoulense) and the ideal gene for species determination. METHODS: Clinical symptoms, laboratory examinations, and radiological examinations were retrospectively reviewed. The hsp65, 16S rRNA, rpoB and ITS region of M. seoulense, were sequenced and phylogenetic trees of mycobacterium strains were constructed. RESULTS: Four M. seoulense strains isolated from 4 patients caused pulmonary infections based on the symptoms and radiological results. The 16S rRNA sequence identified 2 strains as M. intracellulare and the other 2 as Stenotrophomonas maltophilia. In contrast, the rpoB, 16S-23S inter-region (ITS), and hsp65 sequences shared high identity with M. seoulense. Notably, the phylogenetic tree based on the ITS, hsp65, and rpoB sequences clustered 4 M. seoulense strains identified in this study with M. seoulense strains in the database. CONCLUSION: M. seoulense strains can cause infection in humans. They can be identified by sequencing ITS, hsp65, and rpoB genes.

Prevalence of non-tuberculous mycobacteria among previously treated TB patients in the Gulf of Guinea, Africa.

Objective: Differentiation between non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTBC) is crucial for case management with the appropriate antimycobacterials. This study was undertaken in three West and Central African countries to understand NTM associated with pulmonary tuberculosis in the sub-region. Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained from solid and liquid cultures were analysed. The isolates were tested for drug susceptibility, and MTBC were confirmed using IS6110. All IS6110-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.