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Objective: We conducted a cross-sectional study in the five administrative regions of Northern Ghana to determine the diversity of Mycobacterium tuberculosis complex (MTBC) sub/lineages and their susceptibility to isoniazid (INH) and rifampicin (RIF). Methods: Sputum specimens were collected and cultured from 566 pulmonary tuberculosis patients reporting to 17 health facilities from 2015 to 2019. Mycobacterial isolates obtained from solid cultures were confirmed as members of the MTBC by PCR amplification of IS6110 and rpoß and assigned lineages and sub-lineages using spoligotyping. Results: Of 294 mycobacterial isolates recovered, MTBC species identified were: M. tuberculosis sensu stricto (Mtbss) 241 (82.0%), M. africanum 41 (13.9%) and M. bovis four (1.4%) with eight (2.7%) unidentified. The human-adapted lineages (L) identified (N=279) were L1 (8/279, 2.9%), L2 (15/279, 5.4%), L3 (7/279, 2.5%), L4 (208/279, 74.5%), L5 (13/279, 4.7%) and L6 (28/279, 10.0%) with three unidentified lineages. Among the 208 L4, the dominant sub-lineages in the region were the Cameroon 120/208 (57.7%) and Ghana 50/208 (24.0%). We found 4.4% (13/294) and 0.7% (2/294) of the patients infected with MTBC isolates resistant to INH only and RIF only, respectively, with 2.4% (7/294) being infected with MDR strains. Whereas L6 was associated with the elderly, we identified that the Ghana sub-lineage of L4 was associated with both INH and MDR (p

Highlights • Unresolved previous infection as major cause of recurring tuberculosis (TB) in Ghana. • Genomic epidemiology identifies high relapse among recurrent TB cases in Ghana. • 15-locus MIRU-VNTR typing is sufficient to predict the cause of TB recurrence. • Evidence of recent household-related TB transmission in Ghana. • Need for increased education by national TB control program., Objective To retrospectively investigate the cause of recurring tuberculosis (rcTB) among participants with pulmonary TB recruited from a prospective population-based study conducted between July 2012 and December 2015. Methods Mycobacterium tuberculosis complex isolates obtained from rcTB cases were characterized by standard mycobacterial genotyping tools, whole-genome sequencing, and phylogenetic analysis carried out to assess strain relatedness. Results The majority (58.3%, 21/36) of study participants with rcTB episodes had TB recurrence within 12 months post treatment. TB strains with isoniazid (INH) resistance were found in 19.4% (7/36) of participants at the primary episode, of which 29% (2/7) were also rifampicin-resistant. On TB recurrence, an INH-resistant strain was found in a larger proportion of participants, 27.8% (10/36), of which 40% (4/10) were MDR-TB strains. rcTB was attributed to relapse (same strain) in 75.0% (27/36) of participants and 25.0% (9/36) to re-infection. Conclusion Our findings indicate that previous unresolved infectiondue to inadequate treatment, may be the major cause of rcTB.

Objectives: The genetic diversity of Mycobacterium tuberculosis complex (MTBC) influences the immune response of the host, which may affect the immunodiagnostic tests and biomarker assessment studies used for tuberculosis (TB). This study aimed to determine whether the mycobacterial-antigen-stimulated cytokine responses vary with the genotype of the MTBC infecting the patient. Methods: Eighty-one patients with confirmed active pulmonary TB were recruited, and MTBC clinical strains were isolated from their sputum for bacterial lineage single-nucleotide polymorphism typing. Whole blood was drawn from the patients to measure the purified protein derivative (PPD)-stimulated cytokine responses (GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, TNF-α, IFN-α, IL-12, eotaxin, IL-13, IL-15, IL-17, MIP1-α, MIP1-β, MCP1, IL1RA, IP10, IL2R, MIG) with the Luminex multiplex immunoassay. Results: Of the 24 cytokines studied, three were produced differentially in whole blood dependent on the infecting lineage of MTBC. Decreased production of IL-17 was observed in patients infected with modern lineages compared with patients infected with ancestral lineages (P < 0.01), and production of IFN-γ and IL-2 was significantly decreased in patients infected with lineage 4 strains compared with patients infected with lineage 3 strains (P < 0.05). Conclusion: MTBC strains belonging to lineage 4 induced a decreased whole-blood PPD-stimulated pro-inflammatory cytokine response.

The Bacillus Calmette-Guérin (BCG) vaccine is widely used worldwide. Intracranial manifestation as an adverse event of BCG is extremely rare. A previously healthy 16-month-old boy was referred to our hospital for eye contact difficulties and progressive gait disturbance lasting two months. He was inoculated with BCG at seven months of age. Brain magnetic resonance imaging (MRI) revealed hydrocephalus with widespread and disseminated enhancement lesions with thickening of the third ventricle floor, and brain tissue pathologically showed non-caseous granulomatous inflammation. Immunosuppressive therapies were initiated because of a provisional diagnosis of neurosarcoidosis. Three months later, a positive polymerase chain reaction (PCR) result for the Mycobacterium tuberculosis complex was obtained. Eventually, M. bovis (BCG Tokyo 172 strain) was identified in the cerebrospinal fluid (CSF) and shunt tube culture. The prolonged use of antituberculosis drugs and multiple shunt replacement surgeries were needed for recovery. There was no evidence of immunodeficiency. Unfortunately, he had severe neurological sequelae of bilateral blindness and neurodevelopmental delay. Our purpose in this report was to highlight the potential for intracranial manifestations of adverse reactions related to BCG vaccination. We propose that the CSF PCR assay of Mycobacterium tuberculosis (MTB) complex should be applied repeatedly in children suspected of intractable neurosarcoidosis, with a history of BCG vaccination.

Objective: In resource-limited tuberculosis-endemic countries, Mycobacterium tuberculosis in sputum is mainly detected by acid-fast bacillus (AFB) staining and the identification of sputum-derived cultures. PCR techniques are practical only in well-resourced laboratories. This study investigated the application of a rapid, simple, and inexpensive fluorescence in situ hybridization (FISH) assay to identify and differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) in sputum. Methods: The Mycobacterium/Nocardia Genus (MN Genus)-MTBC FISH assay performed in this study utilizes two different DNA probes labeled with different fluorescent molecules that hybridize respectively with 16S rRNA of the genus Mycobacterium and 23S rRNA of MTBC. The assay was tested on 202 patient sputum samples in Mangaluru, Karnataka State, India. Sputa were first liquefied and bacteria concentrated before performing the FISH assay and parallel culturing and AFB staining. The identities of cultured bacteria from DNA sequencing were compared with FISH assay findings from corresponding sputa. Results: Of the 202 sputum samples tested, 67 reacted with both MN Genus-specific and MTBC-specific probes, none reacted only with the MTBC-specific probe, and 22 reacted only with the MN Genus-specific probe. The FISH assay yielded results in 2 h and had a limit of detection of 2.2 × 104 CFU/ml in sputum spiked with cultured M. tuberculosis. The diagnostic sensitivity, specificity, and positive and negative predictive values of the FISH assay for MTBC in patient sputa were 89.7%, 95.5%, 88.0%, and 92.6%, respectively. NTM were a significant cause of tuberculosis-like infections in Mangaluru. Conclusions: The MN Genus-MTBC dual probe fluorescence FISH assay previously applied to cultures can also be utilized in resource-limited tuberculosis-endemic countries for rapidly identifying and differentiating MTBC and NTM in sputum samples. Keywords: Fluorescence in situ hybridization, LED fluorescence microscopy, Mycobacterium tuberculosis, Non-tuberculous mycobacteria, Tuberculosis diagnosis, Sputum assay

Ancient and historical tuberculosis (TB) can be recognized by its typical paleopathology in human remains. Using paleomicrobiology, it is possible to detect many more individuals infected with TB but with no visible lesions. Due to advances in molecular analysis over the past two decades, it is clear that TB was widespread in humans from the Neolithic period and has remained so until the present day. Past human populations were associated with different lineages of the Mycobacterium tuberculosis complex, thereby elucidating early human migrations. Using paleomicrobiology, it is possible to detect individuals infected with TB who are also co-infected with other bacteria or parasites. TB is also found in hosts with co-morbidities such as neoplasms, or metabolic disorders such as rickets and scurvy. In well-preserved human skeletal or mummified tissue, whole genome sequencing has detected individuals with multiple infections of different M. tuberculosis strains. Such studies put modern findings into context and emphasize the importance of human population density in such circumstances.

Tuberculosis (TB) is a major cause of worldwide mortality. We report the case of a non-HIV-infected woman with clinical suspicion of pleural tuberculosis and contradictory results between Xpert® MTB/RIF and Abbott RealTime MTB assays from pleural fluid specimen. Liquid and solid cultures for tuberculosis were performed with negative results. The patient received treatment, and clinical improvement was observed. Both techniques detect Mycobacterium tuberculosis complex, but they have different targets and limits of detection. Abbott RealTime MTB results correlated well with the clinical findings of the patient.

Summary Background The development of novel antibiotics to treat multidrug-resistant (MDR) tuberculosis is time-consuming and expensive. Multiple immune modulators, immune suppressants, anti-inflammatories, and growth enhancers, and vitamins A and D, inhibit Mycobacterium avium subspecies paratuberculosis (MAP) in culture. We studied the culture inhibition of Mycobacterium tuberculosis complex by these agents. Methods Biosafety level two M. tuberculosis complex (ATCC 19015 and ATCC 25177) was studied in radiometric Bactec or MGIT culture. Agents evaluated included clofazimine, methotrexate, 6-mercaptopurine, cyclosporine A, rapamycin, tacrolimus, monensin, and vitamins A and D. Results All the agents mentioned above caused dose-dependent inhibition of the M. tuberculosis complex. There was no inhibition by the anti-inflammatory 5-aminosalicylic acid, which causes bacteriostatic inhibition of MAP. Conclusions We conclude that, at a minimum, studies with virulent M. tuberculosis are indicated with the agents mentioned above, as well as with the thioamide 5-propothiouricil, which has previously been shown to inhibit the M. tuberculosis complex in culture. Our data additionally emphasize the importance of vitamins A and D in treating mycobacterial diseases.

Summary Objective To design and standardize an in-house reverse line blot hybridization (RLBH) assay for the accurate identification of 15 clinically relevant species of mycobacteria and for the detection of drug resistance to rifampin (RIF), isoniazid (INH), and streptomycin (STR) in Mycobacterium tuberculosis complex (MTB). Material and methods Oligonucleotides specific for 15 different species of mycobacteria and wild type and mutant alleles of selected codons in the rpoβ, inhA, katG, rpsL , and rrs genes were designed and immobilized on a membrane. A multiplex PCR was standardized to amplify all target genes. The assay was optimized using ATCC and known mutant strains. Three hundred MTB isolates, 85 non-tuberculous mycobacteria (NTM) isolates, and 48 smear-positive specimens were analyzed. Results were confirmed by PCR restriction enzyme assay and sequencing. Results Upon RLBH analysis, among the NTM, 14% were identified as Mycobacterium fortuitum , 16% were identified as Mycobacterium abscessus , 20% showed 99% homology with Mycobacterium intracellulare , and 31% showed 98% homology with Mycobacterium simiae . Of the 300 MTB isolates analyzed, 75% RIF-resistant isolates had Ser531Leu mutation in the rpoβ gene. Of the INH-resistant isolates, 89% showed Ser315Thr mutation in the katG gene, whereas 16% showed −15 C→T mutation in the promoter region of the inhA gene. Among STR-resistant isolates, 75% had A→G mutation in the rpsL gene at codon 43. RLBH results showed 96–99% concordance with phenotypic culture results. Conclusion This is a first attempt at combining speciation with detection of drug resistance to RIF, INH, and STR in MTB for accurate and rapid management of mycobacterial infections as well as for compiling genotypic epidemiological data.

Background: The rapid identification of mycobacteria from culture-positive Mb/Bact bottles is an important clinical issue. Furthermore, the availability of a non-expensive, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In this study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from positive Mb/Bact (Organon-Teknika) bottles. A multiplex PCR focusing the hsp65 gene, and the IS6110 was done and evaluated for its efficiency compared to PRA identfication, and PNB inhibition. Methods: Mycobacterial strains were grown from clinical samples and preserved in solid media (LJ). A panel of 91 M. tuberculosis strains, and 26 Non-TuberculousMycobacteria were submitted to the following identification techniques: a) hsp65 gene amplification followed by restriction (PRA); b) p-Nitrobenzoic acid (PNB) growth inhibition in liquid media (Mb/Bact); 3) Multiplex-PCR, using two sets of primers: TB284/TB850 (IS6110 MTC spcific primers), and TB11/TB12 (65 kD antigen, Mycobacterium genus specific primers). The mycobacteria was inactivated being heated at 80oC for 20min before DNA extraction. DNA was extracted by the freezing and thawing method. 50 l of the the supernatant was used for amplification. Identification using hsp65 restriction was done according to international protocols (Telenti et al, 1993), and considered the gold-standard. PNB was added to liquid Mb/Bact bottles to a final concentration of 500 g/ml. p-aminobenzoic acid, and the Mycobacterial concentration was adjusted to the MacFarland 1 standard. Results: The hsp65 restriction analysis identified all Mycobacteria tested as: M.tuberculosis (61 strains), M.avium, M.intracellulare, M.fortuitum, M.abscessus, M.gordonae, M.szulgai. There was 100% agreement between the tests performed Conclusion: A simple and inexpensive Multiplex-PCR as described here, can readly distinguish among M. tuberculosis Complex strains, and Non-Tuberculous Mycobacteria.

SummaryCompared to other organs, skin is an uncommon site of tuberculosis involvement. In the era of HIV infection, increased intravenous drug abuse, and the use of immunosuppressive therapy for various systemic diseases, tuberculosis in all its forms, including skin tuberculosis, has reemerged. We report two cases of primary cutaneous tuberculosis in immunocompetent patients and review the literature of all cases described since 1935.

Summary Objective This study represents an early attempt to determine the diversity of Mycobacterium tuberculosis in Egypt, particularly of drug-resistant strains. Methods We characterized 45 Mycobacterium tuberculosis complex isolates from sputum samples of Egyptian patients with pulmonary tuberculosis, in order to establish a database of strain types and antimicrobial susceptibility patterns. Results One Mycobacterium bovis and 44 Mycobacterium tuberculosis (MTB) isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Twenty-five (56.8%) of the 44 MTB isolates were susceptible in vitro to all anti-tuberculosis drugs tested; five (11.4%) were mono-resistant to isoniazid or streptomycin (four were resistant to streptomycin and only one was resistant to isoniazid) and 14 (31.8%) were resistant to more than one drug (multidrug-resistant, MDR). Among the 44 MTB isolates tested by RFLP analysis in this study, 40 different RFLP patterns were obtained. The number of IS6110 copies ranged from 5 to 16. Studying the IS6110 RFLP patterns indicated that the 44 isolates did not cluster together but were generally scattered. None of the 14 MDR isolates were clustered. Twenty-two different spoligotypes were identified among the 44 MTB isolates, of which 13 were unique. The remaining 31 isolates were grouped into nine clusters of strains sharing identical spoligotypes. Conclusions We have demonstrated evidence of diversity among the drug-susceptible and resistant MTB strains. Continued surveillance for strains of MTB involved in pulmonary tuberculosis in Egypt, and especially for drug-resistant strains, is warranted.