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Current tuberculosis (TB) diagnostic tests primarily rely on sputum samples, yet many TB patients cannot produce sputum. This study explored whether saliva could be used instead of sputum to diagnose pulmonary TB (PTB). The study included 32 patients with confirmed PTB and 30 patients with other respiratory diseases (ORD). Saliva from all study participants was subjected to quantitative (qPCR) assays targeting the IS 1081 gene for detection of M. tuberculosis complex DNA. The sensitivity of saliva IS 1081 qPCR was 65.6 % (95 % CI 48.4–80.2 %) with positive results for 21/32 PTB cases, while the specificity was 96.7 % (95 % CI 85.9–99.6 %) with negative results for 29/30 participants with ORD. Sensitivity improved to 72.4 % (95 % CI 54.6–86.0 %) when sputum-Xpert was used as the reference standard, while remaining similar at 65.5 % (95 % CI 47.4–80.7 %) when culture was used as the reference standard. In receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) for saliva IS 1081 qPCR was 82.5 % (95 % CI 71.7–93.3 %). Saliva testing offers a promising alternative to sputum for TB diagnosis among confirmed PTB cases. Larger multicenter studies, encompassing diverse clinical TB characteristics, are needed to provide improved estimates of diagnostic sensitivity and specificity. [ABSTRACT FROM AUTHOR]

The genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains was characterized among isolates from individuals with pulmonary tuberculosis (PTB) symptoms attended holy water sites (HWSs) in the Amhara region, Ethiopia. A cross-sectional study was done from June 2019 to March 2020 to describe the genetic diversity and drug-resistance profiles of MTBC isolates. Sputum specimens were collected and cultured in the Löwenstein-Jensen culture medium. Line Probe Assay, MTBDR plus VER 2.0, and MTBDR sl VER 2.0 were used to detect first-and second-line anti-TB drug-resistance patterns. A spoligotyping technique was utilized to characterize the genetic diversity. Statistical analysis was performed using STATA 15. Of 560 PTB-symptomatic participants, 122 (21.8%) were culture-positive cases. Spoligotyping of 116 isolates revealed diverse MTBC sublineages, with four major lineages: Euro-American (EA) (Lineage 4), East-African-Indian (EAI) (Lineage 3), Ethiopian (ETH) (Lineage 7), East Asian (EA) (Lineage 2). The majority (96.6%) of the isolates were EA (lineage 4) and EAI, with proportions of 54.3% and 42.2%, respectively. A total of 31 spoligotype patterns were identified, 26 of which were documented in the SITVIT2 database. Of these, there were 15 unique spoligotypes, while eleven were grouped with 2-17 isolates. SIT149/T3-ETH (n = 17), SIT26/CAS1-DELHI (n = 16), SIT25/CAS1-DELHI (n = 12), and SIT52/T2 (n = 11) spoligotypes were predominant. A rare spoligotype pattern: SIT41/Turkey and SIT1/Beijing, has also been identified in North Shewa. The overall clustering rate of sub-lineages with known SIT was 76.4%. Of the 122 culture-positive isolates tested, 16.4% were resistant to rifampicin (RIF) and/or isoniazid (INH). Multidrug-resistant TB (MDR-TB) was detected in 12.3% of isolates, five of which were fluoroquinolones (FLQs) resistant. SIT149/T3-ETH and SIT21/CAS1-KILI sublineages showed a higher proportion of drug resistance. Diverse MTBC spoligotypes were identified, with the T and CAS families and EA (lineage 4) predominating. A high prevalence of drug-resistant TB, with SIT149/T3-ETH and CAS1-KILI sublineages comprising a greater share, was observed. A study with large sample size and a sequencing method with stronger discriminatory power is warranted to understand better the genetic diversity of circulating MTBC in this cohort of study, which would help to adopt targeted interventions. • SIT149/T3-ETH (n = 17), SIT26/CAS1-DELHI (n = 16), and SIT25/CAS1-DELHI (n = 12) spoligotypes were predominant. • A rare spoligotype pattern: SIT41/Turkey and SIT1/Beijing, has also been identified in North Shewa. • SIT149/T3-ETH and SIT21/CAS1-KILI sublineages showed a higher proportion of drug resistance. [ABSTRACT FROM AUTHOR]

Tuberculosis, caused by Mycobacterium tuberculosis complex, is a leading cause of mortality in the world, and 15% of the patients may present with extrapulmonary diseases, including splenic lesion. However, isolated splenic infection with M. tuberculosis complex is very rare. A 19-year-old otherwise healthy woman presented with left flank pain, revealing FDG-avid nodules in the spleen. She did not have pulmonary lesions. Histopathology of splenectomized sample showed granuloma, and subsequent PCR revealed amplification of IS 6110 , a genetic sequence exclusively detected in M. tuberculosis complex. A wide range of differential diagnosis of isolated splenic lesion should include M. tuberculosis infection regardless of pulmonary involvement. An elective splenectomy may be mandatory in timely manner. [ABSTRACT FROM AUTHOR]

Lineage 1 (L1) is one of seven Mycobacterium tuberculosis complex (MTBC) lineages. The objective of this study was to improve the complex taxonomy of L1 using phylogenetic SNPs, and to look for the origin of the main L1 sublineage prevalent in Para, Brazil. We developed a high-throughput SNPs-typing assay based on 12-L1-specific SNPs. This assay allowed us to experimentally retrieve SNP patterns on nine of these twelve SNPs in 277 isolates previously tentatively assigned to L1 spoligotyping-based sublineages. Three collections were used: Pará-Brazil (71); RIVM, the Netherlands (102), Madagascar (104). One-hundred more results were generated in Silico using the PolyTB database. Based on the final SNPs combination, the samples were classified into 11 clusters (C1-C11). Most isolates within a SNP-based cluster shared a mutual spoligotyping-defined lineage. However, L1/EAI1-SOM (SIT48) and L1/EAI6-BGD1 (SIT591) showed a poor correlation with SNP data and are not monophyletic. L1/EAI8-MDG and L1/EAI3-IND belonged to C5; this result suggests that they share a common ancestor. L1.1.3/SIT129, a spoligotype pattern found in SNPs-cluster C6, was found to be shared between Pará/Brazil and Malawi. SIT129 was independently found to be highly prevalent in Mozambique, which suggests a migration history from East-Africa to Brazil during the 16th–18th slave trade period to Northern Brazil. • M. tuberculosis L1 has a good correlation between SNP phylogeny and spoligotyping. EAI8_MDG and EAI3_IND, L1.1.2, are evolutionary linked. • EAI8_MDG and EAI3_IND, L1.1.2, are evolutionary linked. • SIT129 and SNPs profile suggest a shared evolutionary history between Pará-Brazil, Malawi and Mozambique. [ABSTRACT FROM AUTHOR]

Two strains of Mycobacterium tuberculosis complex can be separated as much as 2500 single nucleotide differences (Coscolla and Gagneux, 2014). In that limited amount of diversity, we find an astonishing range of clinical, epidemiological and biological phenotypes. The most striking is the strong host preferences depending on the infecting strain while more subtle differences can be found looking at different human tuberculosis isolates. Those subtle differences are the most difficult to spot given that analysis methods for so little diversity are limited and phenotypes like virulence are difficult to define and measure. Recent genomics advances allow to study the pathogen diversity at a resolution not available before from comparative species level, to global diversity to transmission in local settings. Here, we will review some of these recent results to highlight how population genomics approaches can aid not only to understand how MTBC evolved but also to identify relevant biomedical targets. [ABSTRACT FROM AUTHOR]

Whole T cell interferon gamma release assays such as QuantiFERON-TB Gold Plus (QTF-TB) are used to evaluate Mycobacterium tuberculosis complex (MTC) exposure but fail to discriminate latent tuberculosis infection (LTBI) from active disease. In this study conducted in a low-burden area, 1215 patients presenting MTC risk and tested both for QTF-TB and mycobacterial infection (microscopy, culture, and/or PCR) were selected, as well as 1298 controls screened with QTF-TB before medical recruitment. The humoral response (LIODetect®TB-ST) was further evaluated in 199 selected patients. In patients with active disease, MTC positivity (culture and/or PCR with species identification) was associated with QTF-TB positivity (45/56, 80.4 %). Although QTF-TB1/TB2 peptides were not suitable for discriminating against active MTC disease from LTBI, the cut-off value of 4.4 IFN-γ IU/mL produced the best diagnostic performance for MTC detection. Lower levels of QTF-TB were reported among patients with isolated active pulmonary MTC as compared to a lymph-nodal location and a disseminated form. Next, antibodies were detected in 4/55 (7.3 %) active MTC disease cases, while negative in cases of LTBI and indeterminate/negative QTF-TB. In conclusion, the added value to combine cellular (QTF-TB) and humoral (LIODetect®TB-ST) assays to predict an active MTC disease is limited. • Mycobacteriological and QTF-TB positivity overlap imperfectly in a low-burden area. • TB location site and immunosuppression status affect QTF-TB levels. • Coupling cellular and humoral responses are limited in TB diagnosis. [ABSTRACT FROM AUTHOR]

Currently, pulmonary tuberculosis (TB) isolation recommendations are based on serial sputum smear microscopy. To assess infectiousness of smear-negative/GeneXpert-positive (Sm-/GXpert+) pulmonary TB, we evaluated 511 contacts of pulmonary TB patients attended at a teaching hospital in Spain (2010-2018). There were no statistically significant differences in rates of Mycobacterium tuberculosis infection (46.2% contacts of smear-positive and 34.6% contacts of Sm-/GXpert+ pulmonary TB patients, p = 0.112). Sm-/GXpert+ pulmonary TB poses a substantial risk of transmission of M. tuberculosis infection. Our results add evidence to support including Real-time Polymerase Chain Reaction (XpertⓇMTB/RIF) in the work-up diagnosis of suspected pulmonary TB cases to make decisions on air-borne isolation. [ABSTRACT FROM AUTHOR]

Highlights • Both ELISAs provide 100% specificity for TB diagnosis in intensive-managed pigs. • P22 ELISA had better specificity and sensitivity than bPPD ELISA in free-ranging suids. • TB sero-diagnostic tests are valuable tools in the control of the disease in suids. Abstract Background and objectives Animal tuberculosis (TB) is a multi-host disease involving a wide variety of domestic and wild mammals and causing a significant economic burden and sanitary problems. Wild boar and domestic pigs (Sus scrofa) are indicators of the circulation of the Mycobacterium tuberculosis complex (MTC) and can play a role in its maintenance. The proper diagnosis of MTC contact in these species is, therefore, a key factor as regards controlling TB. The objective of the current study is to evaluate the diagnostic performance of the protein complex P22 as a candidate for use in an in-house ELISA to identify M. tuberculosis complex-specific antibodies for the diagnosis of TB in comparison to the commonly used bPPD-based ELISA (bPPD ELISA) in suids. Methods We conducted a retrospective study. Sera were collected from wild boar during hunting season and from domestic pigs during routine handling, and all the animals underwent reference standard tests (detailed necropsy followed by bacteriological culture and isolation). Animal TB was confirmed to be positive in 277 animals and negative in 366 animals based on both reference standard tests. Sera from those animals were tested by P22 ELISA as well as bPPD ELISA. Results Both ELISAs yielded a good diagnostic value, however, a higher sensitivity (Se) and specificity (Sp) was achieved with the P22 ELISA (Se: 84.1%; CI 95% : 79.3–88.2% / Sp: 98.4%; CI 95% :96.5–99.4%) when compared to the bPPD ELISA (Se: 77.3%; CI 95% : 71.9–82.2% / Sp: 97.3%; CI 95% : 95–98.3%). An optimum Sp of 100% (CI 95% : 98.54–100%) was attained with white pigs for both the bPPD and the P22 ELISA. Discussion The results suggest that serological tests for MTC-antibody detection, and particularly the P22 ELISA, are valuable tools in the diagnosis of TB in wild boar and domestic pigs when attempting to detect contact with MTC and thereby facilitate TB control and management. [ABSTRACT FROM AUTHOR]

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is an infectious disease with more than 10.4 million cases and 1.7 million deaths reported worldwide in 2016. The classical methods for detection and differentiation of mycobacteria are: acid-fast microscopy (Ziehl-Neelsen staining), culture, and biochemical methods. However, the microbial phenotypic characterization is time-consuming and laborious. Thus, fast, easy, and sensitive nucleic acid amplification tests (NAATs) have been developed based on specific DNA markers, which are commercially available for TB diagnosis. Despite these developments, the disease remains uncontrollable. The identification and differentiation among MTBC members with the use of NAATs remains challenging due, among other factors, to the high degree of homology within the members and mutations, which hinders the identification of specific target sequences in the genome with potential impact in the diagnosis and treatment outcomes. In silico methods provide predictive identification of many new target genes/fragments/regions that can specifically be used to identify species/strains, which have not been fully explored. This review focused on DNA markers useful for MTBC detection, species identification and antibiotic resistance determination. The use of DNA targets with new technological approaches will help to develop NAATs applicable to all levels of the health system, mainly in low resource areas, which urgently need customized methods to their specific conditions. [ABSTRACT FROM AUTHOR]

We examined categorical agreement between automated mycobacterial susceptibility testing methods (Mycobacterial Growth Indicator Tube [MGIT] 960 System and the VersaTREK Mycobacteria Detection and Susceptibility System) which are based on single critical concentration (CC) “breakpoints” and a commercial microbroth dilution method (Sensititre Mycobacterium tuberculosis MIC Plate [MYCOTB]) which provides an MIC value. Mycobacterium tuberculosis isolates (n=355) were tested against three first-line antimycobacterial agents (ethambutol [EMB], isoniazid [INH], rifampin [RIF]) using the MYCOTB plate and either the MGIT 960 (site 1, n=142) or VersaTREK (site 2, n=213) systems. Overall categorical agreement was 96.8%. When stratified by drug and CC-defined susceptible and resistant isolates, concordance ranged from 75% to 100%. Interpretation of MIC-based results versus established CC-based results was challenging for drugs whose CC was not represented by an exactly equivalent concentration in the manufacturer-defined dilutions on the MYCOTB plate (EMB, INH). We propose interpretations of MYCOTB plate MICs using the currently available plate configuration. [ABSTRACT FROM AUTHOR]

This retrospective study covered over two decades, during which an individual head and neck surgeon treated 24 patients with cervicofacial lymphadenitis that was related to both Mycobacterium tuberculosis complex (n = 17, made up of M tuberculosis (n = 16) and M bovis (n = 1)), and non-tuberculous mycobacteria. The seven cases of non-tuberculous mycobacteria were caused by M avium complex (n = 3), M malmoense (n = 3), and M kansaii (n = 1). By using a tailored management approach, at times selective combined surgical and antimycobacterial treatment, he achieved a success rate of 23/24 cases, with only one recurrence and no major complications. The results suggest that patients with tuberculosis confined to the head and neck rarely develop constitutional symptoms, so the absence of such symptoms may not exclude tuberculosis. There was also a good correlation between predictive variables (immune state, inflammatory markers on admission, causative mycobacterium, and the antimycobacterial regimen used) and time spent under follow-up at the head and neck outpatient clinic. [ABSTRACT FROM AUTHOR]

Rapid diagnosis of Mycobacterium tuberculosis complex (MTBC) is crucial for the control and treatment of tuberculosis. Currently available diagnostic tools are unable to meet clinical needs, especially in resource limited settings, as they suffer from being time-consuming, costly, insensitive, or dependant on sophisticated instruments and well trained personnel. An optical diagnostic platform (SPMS-AIA) consisting of the silicon photonic microring sensor and the asymmetric isothermal amplification technique was developed to detect MTBC in a rapid (one hour), isothermal, label-free and real-time manner. The performance of the SPMS-AIA platform was assessed by detecting two MTBC specific biomarkers, IS 6110 and IS 1081 . Detection limits were as low as 3.2 copies for IS 6110 and 12 copies for IS1081 respectively when tested with Mycobacterium tuberculosis H37Rv genomic DNA. An evaluation for detection of IS 6110 in clinical sputa showed that the SPMS-AIA platform performed as well as the established market leader GeneXpert MTB/RIF platform. Of 26 MTBC culture-positive and smear-positive specimens, 23 were found positive by the SPMS-AIA platform (89% sensitivity). Of 47 MTBC culture-positive and smear-negative specimens, 34 were positive by the SPMS-AIA platform (72% sensitivity): this is similar to the sensitivity of 70% by the GeneXpert MTB/RIF platform. The versatility of the SPMS-AIA platform was demonstrated by multiplex detection of IS 6110 and IS 1081 in 10 clinical specimens. The SPMS-AIA platform shows great potential for development into a point-of-care (POC) diagnostic tool for use in resource limited settings. [ABSTRACT FROM AUTHOR]

Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3′-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers. [ABSTRACT FROM AUTHOR]

Résumé Mycobacterium canettii est une bactérie du complexe tuberculosis isolée pour la première fois par Georges Canetti en 1969. Sa particularité est l’aspect lisse des colonies en culture, la distinguant des autres espèces du complexe tuberculosis. Par ailleurs, elle présente une grande diversité génétique, bien supérieure à celle de M. tuberculosis . Les récentes études de génomique placent M. canettii comme l’ancêtre commun des mycobactéries. Contrairement à M. tuberculosis , le réservoir et le mode de transmission de M. canettii restent totalement inconnus et l’hypothèse d’un réservoir environnemental est la plus souvent évoquée. En 2015, on recense une centaine de souches de M. canettii dans la littérature. Les descriptions cliniques d’infections à M. canettii sont dominées par les localisations pulmonaires (52 %) et extra thoraciques avec un fort tropisme ganglionnaire (32 %) essentiellement observées chez des patients ayant séjourné dans la Corne de l’Afrique, en particulier à Djibouti. Les arguments cliniques, radiologiques et histologiques ne sont pas spécifiques de la tuberculose et le diagnostic de certitude repose sur la mise en évidence de M. canettii dans les prélèvements. Les techniques de détection moléculaire réalisées directement sur prélèvements ne permettent que le diagnostic du complexe tuberculosis . C’est la culture qui permet d’identifier M. canettii précisément et fournit le matériel nécessaire à la réalisation de l’antibiogramme. L’identification peut se faire par des caractères phénotypiques mais de plus en plus moléculaires ou par spectrométrie de masse. Le traitement est identique à celui de M. tuberculosis , aucun cas de résistance n’ayant été décrit dans la littérature. Mycobacterium canettii is a bacillus related to the tuberculosis complex isolated for the first time by Georges Canetti in 1969. Its particularly is the smooth appearance of the colonies in culture, distinguishing it from the other species of the tuberculosis complex. In addition, it has a great genetic diversity, much higher than that of M. tuberculosis . Recent genome studies place M. canettii as the common ancestor of mycobacteria. Unlike M. tuberculosis , the reservoir and way of transmission of M. canettii remain totally unknown and the hypothesis of an environmental reservoir is most often mentioned. By 2015, there are about 100 strains of M. canettii in the literature. Clinical descriptions of M. canettii infections are dominated by pulmonary (52%) and extra-thoracic sites with a high lymph node tropism (32%), mainly observed in patients who have stayed in the Horn of Africa, particularly Djibouti. The clinical, radiological and histological arguments are not specific to tuberculosis and the diagnosis is based on the detection of M. canettii in the samples. Molecular detection techniques performed directly on specimens only allow detection of the tuberculosis complex. The culture is the main way to identify M. canettii precisely and allows antibiotic susceptibility testing. The identification can be done by phenotypic characters but more and more molecular or by mass spectrometry. The treatment is identical to that of M. tuberculosis , no case of resistance are described. [ABSTRACT FROM AUTHOR]

Background The differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. Methods A total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. Results The sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15–100%, p < 0.0001) and 97.8% (95% CI 91.86–99.87%, p < 0.0001), respectively. The results of conventional drug susceptibility testing and the QMAP Dual-ID assay were completely concordant for all clinical isolates (100%, 95% CI 98.56–100%). Out of 223 M. tuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations ( n = 26, 96.3%), including those missing wild-type probe signals, were located in three codons (529–534, 524–529, and 514–520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). Conclusions The entire QMAP system assay takes about 3 h to complete, while results from the culture-based conventional method can take up to 48–72 h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples. [ABSTRACT FROM AUTHOR]

Rapid and reliable diagnosis is crucial for correct management of tuberculosis. The Abbott RealTi m e MTB Assay represents a novel qualitative real-time PCR assay for direct detection of M. tuberculosis -complex (MTB) DNA from respiratory samples. The test targets two highly conserved sequences, the multi-copy insertion element IS 6110 and the protein antigen B (PAB) gene of MTB, allowing even the detection of IS 6610 -deficient strains. We evaluated this commercial diagnostic test by analyzing 200 respiratory and, for the first time, 87 non-respiratory clinical specimens from our tertiary care institution and compared its results to our IS 6110 -based in-house real-time PCR for MTB as well as MTB culture. Overall sensitivity for Abbott RealTi me MTB was 100% (19/19) in smear positive and 87.5% (7/8) in smear negative specimens, while the specificity of the assay was 100% (260/260). For both non-respiratory smear positive and smear negative specimens Abbott RealTi me MTB tests showed 100% (8/8) sensitivity and 100% (8/8) specificity. Cycle threshold (Ct) value analysis of 16 MTB positive samples showed a slightly higher Ct value of the Abbott RealTi me MTB test compared to our in-house MTB assay (mean delta Ct = 2.55). In conclusion, the performance of the new Abbott RealTi me MTB Assay was highly similar to culture and in-house MTB PCR. We document successful analysis of 87 non-respiratory samples with the highly automated Abbott RealTi m e MTB test with no inhibition observed. [ABSTRACT FROM AUTHOR]

Summary Mycobacterium africanum comprises two phylogenetic lineages within the Mycobacterium tuberculosis complex (MTBC). M. africanum was first described and isolated in 1968 from the sputum of a Senegalese patient with pulmonary tuberculosis (TB) and it has been identified increasingly as an important cause of human TB, particularly prevalent in West Africa. The restricted geographical distribution of M. africanum , in contrast to the widespread global distribution of other species of MTBC, requires explanation. Available data indicate that M. africanum may also have important differences in transmission, pathogenesis, and host–pathogen interactions, which could affect the evaluation of new TB intervention tools (diagnostics and vaccines)–those currently in use and those under development. The unequal geographical distribution and spread of MTBC species means that individual research findings from one country or region cannot be generalized across the continent. Thus, generalizing data from previous and ongoing research studies on MTBC may be inaccurate and inappropriate. A major rethink is required regarding the design and structure of future clinical trials of new interventions. The West, Central, East, and Southern African EDCTP Networks of Excellence provide opportunities to take forward these pan-Africa studies. More investments into molecular, epidemiological, clinical, diagnostic, and immunological studies across the African continent are required to enable further understanding of host– M. africanum interactions, leading to the development of more specific diagnostics, biomarkers, host-directed therapies, and vaccines for TB. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a multi-host infectious disease caused by members of the Mycobacterium tuberculosis complex (MTC). In Mediterranean ecosystems, where multiple animal hosts of TB are present, identifying the role of the different species involved in the epidemiology of TB is a key point to be able to implement proper control measures. Sheep are susceptible to MTC infection but have traditionally been considered a spillover host. However, the occurrence of outbreaks involving sheep in recent years evidences the need to better understand the role of this small ruminant species in the epidemiology of the disease. Here, we aimed to determine the seroprevalence and risk factors associated with MTC seropositivity in sheep in Andalusia (southern Spain), a region with one of the highest prevalence of MTC infection in both cattle and wild ungulates. A total of 2266 sheep from 83 flocks were tested for antibodies against MTC using an in-house indirect ELISA. Anti-MTC antibodies were detected in 16 (0.7%) of the 2266 sheep (adjusted true prevalence 0.29%, 95% posterior probability interval 0.01–1.05). Seropositivity was found in 14.5% (12/83; 95%CI: 6.9–22.0) of the sheep farms analyzed. A semi-extensive management system was identified as a risk factor associated with MTC seropositivity in sheep farms (OR = 3.7; p < 0.038; 95%CI: 1.1–12.4) in the study area. To the best of the authors' knowledge, this is the first active TB surveillance study carried out to assess MTC exposure in sheep. Our results indicate MTC circulation in sheep farms in southern Spain. However, the low individual seroprevalence obtained suggests that sheep may play a limited role in the epidemiology of TB in this region. Serosurveillance programs could be a valuable tool to detect MTC circulation in sheep in risk scenarios or target farms, in order to optimize control measures on TB animal in multi-host Mediterranean ecosystems. • First active TB surveillance study carried out to assess MTC exposure in sheep. • Our results confirm the circulation of MTC in sheep farms in southern Spain. • Semi-extensive management was identified as a risk factor associated with MTC seropositivity. • Sheep may play a limited role in the epidemiology of TB in southern Spain. [ABSTRACT FROM AUTHOR]

Multidrug-resistant (MDR) or extensively drug-resistant (XDR) Tuberculosis (TB) is a major challenge to global TB control. Therefore, accurate tracing of in-country MDR-TB transmission are crucial for the development of optimal TB management strategies. This study aimed to investigate the diversity of MTBC in Nigeria. The lineage and drug-resistance patterns of the clinical MTBC isolates of TB patients in Southwestern region of Nigeria were determined using the WGS approach. The phenotypic DST of the isolates was determined for nine anti-TB drugs. The sequencing achieved average genome coverage of 65.99X. The most represented lineages were L4 (n = 52, 83%), L1 (n = 8, 12%), L2 (n = 2, 3%) and L5 (n = 1, 2%), suggesting a diversified MTB population. In term of detection of M/XDR-TB, while mutations in katG and rpoB genes are the strong predictors for the presence of M/XDR-TB, the current study also found the lack of good genetic markers for drug resistance amongst the MTBC in Nigeria which may pose greater problems on local tuberculosis management efforts. This high-resolution molecular epidemiological data provides valuable insights into the mechanistic for M/XDR TB in Lagos, Nigeria. [ABSTRACT FROM AUTHOR]

The article discusses the use of accurate and affordable antimicrobial susceptibility testing (AST) to control tuberculosis. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that MIC methods are needed in some routine clinical scenarios wherein it can resolve discordances between genotypic and phenotypic AST results for resistance mechanisms with MIC distributions that are divided by a clinical breakpoint.

A novel strip array was developed for a nine-spacer spoligotyping scheme of Mycobacterium tuberculosis complex (MTBC). The new method was evaluated using 211 MTBC isolates and the results were fully concordant with the traditional spoligotyping approach. The strip array proved to be rapid and convenient for spoligotyping of MTBC. [ABSTRACT FROM AUTHOR]

Objective We aimed to evaluate the GeneXpert ® MTB/RIF test for the diagnosis of extrapulmonary tuberculosis. The test simultaneously detects Mycobacterium tuberculosis complex and resistance to rifampicin. Methods We analyzed 153 clinical samples collected in a tertiary hospital in Sfax, Tunisia, between 2013 and 2014. We performed the GeneXpert ® test, a Ziehl-Neelsen and auramine-rhodamine staining, conventional culture on MGIT 960 and LJ media, and we tested the resistance to anti-tuberculosis drugs on MGIT 960 and LJ media for each sample. Diagnosis was based on clinical, radiological, microbiological, pathological, and therapeutic data. Results We considered that 59 patients out of 153 presented with tuberculosis. PCR was positive in 50 samples and all of these samples were susceptible to rifampicin. Sensitivity, specificity, positive predictive value, and negative predictive value of the GeneXpert ® test were 84.7%, 96.8%, 94.3%, and 91%, respectively, compared with diagnosis. We observed a statistically significant difference between the direct test and the GeneXpert ® test, and between culture and the GeneXpert ® test. No statistically significant difference was observed between pathological results and the GeneXpert ® test. Sensitivity of the GeneXpert ® test was 87.5% in biopsies, 80% in pus and abscesses, and 66.7% in biological fluids. All strains were susceptible to rifampicin with culture and GeneXpert ® test. Conclusion The GeneXpert ® test helped detect a higher proportion of M. tuberculosis complex. It does not replace conventional diagnostic methods but it is a useful addition to achieve better sensitivity and obtain rapid results. [ABSTRACT FROM AUTHOR]

The contribution of wildlife species to pathogen maintenance in multi-host communities has seldom been quantified. To assess the relative contribution of the main wildlife hosts of animal tuberculosis (TB) to its maintenance, we estimated the basic reproduction number (R 0) of Mycobacterium tuberculosis complex in wild boar and red deer at 29 sites in the Iberian Peninsula. Host abundance and true TB prevalence were estimated for each species at each site by sampling from distributions incorporating the uncertainty in the proportion of the population harvested each year, sensitivity, and specificity of the diagnostic methods, while excretion of mycobacteria was estimated using site-occupancy models. The distributions of these parameters were then used to estimate, at each site, the R 0,wild boar (range 0.1 – 55.9, average 8.7, standard deviation 11.8), and the R 0,red deer (0.1 – 18.9, 2.2, 3.9). Animal TB is maintained in epidemiological scenarios ranging from any single species acting as a maintenance host (the wild boar in 18 sites and the red deer in 5), to facultative multi-host disease (6 sites). The prevalence of TB in the red deer is likely an important driver of the epidemiology in multi-host communities. The wild boar was the main maintenance host of TB in most of the study sites and could have an epidemiological role linking the wildlife multi-host community and livestock. • Mycobacterium tuberculosis complex circulates in multi-host wildlife communities. • Animal tuberculosis is maintained by single host species or as a multi-host disease. • The wild boar is the main maintenance host in the Iberian Peninsula. • The red deer is an important driver of the multi-host epidemiology. • The wild boar could act as bridge host linking wildlife communities and livestock. [ABSTRACT FROM AUTHOR]

The P22 ELISA was recently developed for the serodiagnosis of animal tuberculosis. Herein, the stability of the P22 antigen in different presentations and storage conditions, and the cross-reactivity with Corynebacterium pseudotuberculosis infection in small ruminants were evaluated. For the stability assay, serum samples from cows, sheep, goats, alpacas, badgers, and wild boar were used in the P22 ELISA. The cross-reactivity analysis used sera from sheep and goats with caseous lymphadenitis (CLA). Differences in the immune recognition of P22 were found when the antigen was stored at 40 °C, but without altering the negative or positive status of each sample. P22 ELISA presented 5.71 % cross-reactivity when CLA-positive sheep were evaluated, but no cross-reaction was observed among CLA-positive goat serum samples. This study showed that the P22 protein complex is stable under different formulations and temperatures, and that the assay presents a low cross-reactivity with CLA. • P22 ELISA is a very stable diagnostic tool for tuberculosis diagnosis. • P22 protein complex is more stable when maintained in solution at 4 °C. • Freeze-drying is a reliable tool for P22 storage. • P22 ELISA did not present cross-reactions for CLA-positive goats. • P22 ELISA presented a 5.7 % cross-reactivity for CLA-positive sheep. [ABSTRACT FROM AUTHOR]

Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar ( Sus scrofa ). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2–6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture ( n = 53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2–6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis -infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis , a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis. [ABSTRACT FROM AUTHOR]

In our laboratory, Mycobacterium caprae has poor growth in standard medium (SM) 7H9-OADC supplemented with pyruvate and Tween-80. Our objectives were to identify mutations affecting M. caprae metabolism and use this information to design a culture medium to improve its growth. We selected 77 M. caprae genomes and sequenced M. caprae NLA000201913 used in our experiments. Mutations present in >95% of the strains compared to Mycobacterium tuberculosis H37Rv were analyzed in silico for their deleterious effects on proteins of metabolic pathways. Apart from the known defect in the pyruvate kinase, M. caprae has important lesions in enzymes of the TCA cycle, methylmalonyl cycle, B 12 metabolism, and electron-transport chain. We provide evidence of enzymatic redundancy elimination and epistatic mutations, and possible production of toxic metabolites hindering M. caprae growth in vitro. A newly designed SM supplemented with l -glutamate allowed faster growth and increased final microbial mass of M. caprae. However, possible accumulation of metabolic waste-products and/or nutritional limitations halted M. caprae growth prior to a M. tuberculosis -like stationary phase. Our findings suggest that M. caprae relies on GABA and/or glyoxylate shunts for in vitro growth in routine media. The newly developed medium will improve experiments with this bacterium by allowing faster growth in vitro. • Members of the Mycobacterium tuberculosis complex occupy distinct ecological niches that determine their metabolic features. • Mycobacterium caprae has poor growth in standard culture medium compared to M. tuberculosis , hampering the research • Results suggest M. caprae eliminated redundant enzymes and depends on GABA and/or glyoxylate shunts for in vitro growth. • Growth halt of M. caprae is due to metabolic waste-products or nutrient limitation resulting from lesions in the metabolism • A newly designed culture medium increased the replication rate of M. caprae. [ABSTRACT FROM AUTHOR]

Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of tuberculosis (TB) in both humans and animals. MTC species are genetically very similar but may differ in their epidemiology, namely geographic distribution and host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species of the MTC. In this work we describe a rapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals. The first step allows the confirmation of the cultures as MTC members, by targeting their IS 6110 element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows the assessment of the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows us to infer the species of the isolate as M. tuberculosis (if all RDs are present), Mycobacterium caprae (if only RD1 and RD4 are present) and Mycobacterium bovis (if only RD1 is present). The identification algorithm developed presented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970 (CI P95% 0.929–1.000). The assay is able to be adaptable to automation and implementation in the routine diagnostic framework of veterinary diagnostic laboratories, with a particular focus for reference laboratories. [ABSTRACT FROM AUTHOR]

Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species. [ABSTRACT FROM AUTHOR]

Abstract: There are few commercial assays that easily and correctly identify the mycobacteria from culture in a clinical laboratory with a high workload. Thus, we developed and evaluated a scheme for the identification of mycobacteria using a multiplex real-time PCR assay and report on its application in our laboratory. The scheme consisted of 3 stepwise PCRs. Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) were differentially detected in the step 1 PCR, and the NTM species were identified in the step 2 and 3 PCRs. Over the 1.5-year study period, 1136 isolates of MTC and 618 isolates of NTM were detected, and the species of 608 (98.4%) of the 618 NTM isolates were identified. We conclude that the established scheme is a very useful diagnostic approach for the rapid and accurate identification of MTC and clinically relevant NTM in a clinical laboratory in a region where tuberculosis is endemic. [Copyright &y& Elsevier]

Abstract: We developed, evaluated, and implemented a Taqman multiplex real-time polymerase chain reaction (PCR) assay for the detection of Mycobacterium avium complex (MAC), targeting the 16S-23S rRNA internal transcribed spacer, which we have combined with an existing Mycobacterium tuberculosis complex assay for use directly in clinical respiratory specimens. Evaluation of the performance of this assay for MAC detection included 464 clinical respiratory specimens tested prospectively. This real-time PCR assay was found overall to have a sensitivity of 71.1%, a specificity of 99.5%, a positive predictive value of 98.0%, and a negative predictive value of 90.2% for MAC. The assay provides results prior to the availability of cultured material and identification, most within 24 h of specimen receipt, and may reduce the need to culture MAC-PCR–positive specimens when susceptibility testing is not requested. Additionally, we have found significant cost savings of approximately $21.00 per specimen and staff time reductions of 3.75 h per specimen with implementation of this assay. [Copyright &y& Elsevier]

Abstract: Upconversion nanoparticles (UCNPs), which are excited at near-infrared wavelength (980nm), emit high-energy photons. Since UCNPs display a high signal-to-noise ratio and no photobleaching, they are extremely useful for diagnostic application. In this study, we applied UCNPs for detecting the IS6110 sequence of the Mycobacterium tuberculosis complex (MTBC) and evaluated the feasibility of the system for use in molecular diagnostics. Using biotinylated primers, IS6110 DNA PCR was performed and the PCR amplicon was then mixed with streptavidin-conjugated UCNPs, followed by intercalation with SYTOX Orange dye. Fluorescence detection for the Förster resonance energy transfer (FRET) of the UCNPs (UCNP-FRET) was then performed. The estimated lowest detection by UCNP-FRET was 102 copies/μL of IS6110 DNA (157bp). The kappa agreement of the UCNP-FRET assay with conventional PCR was 0.8464 (95% confidence interval, 0.7442–0.9486) and false-negative results were reduced. Our results demonstrated the successful implementation of the UCNP-FRET system in detecting the IS6110 sequence of the MTBC and its potential application for molecular diagnostics. [Copyright &y& Elsevier]

Drug resistant tuberculosis (DR-TB) is an important public health issue in different parts of the world. Mycobacterium tuberculosis complex variants (MTBC vars) preferentially infect certain hosts, limiting their distribution to different ecosystems. However, MTBC vars can infect other hosts beyond their preferred target potentially contributing to persistence of drug resistance (DR) in other niches. Here, we performed a comprehensive intra-host genetic analysis for the identification of DR-related mutations among all MTBC minor vars whole genome sequences (8,095 strains) publicly available worldwide. High confidence drug-resistance mutations in kat G (isoniazid), rps L (streptomycin), pnc A (pyrazinamide), rpo B (rifampicin) and gyr A (fluoroquinolones) genes were identified among intrahost minor sub-populations in 197 different strains (2.43%) belonging to vars africanum, bovis, caprae, microti, orygis and pinnipedii. In addition, a three-dimensional structure modeling analysis to assess the role of novel mutations was also performed. Our findings highlight the importance of detecting discrete intra-host populations carrying DR mutations. • MTBC minor variants infecting non-human hosts harbor drug resistance mutations. • Intrahost bacterial populations aid drug resistance mutation emergence and persistence. • Insufficient sequence depth and inadequate algorithms contribute to neglect the existence of drug resistance mutations. [ABSTRACT FROM AUTHOR]

Summary: Mycobacterium tuberculosis is the major causative agent of tuberculosis in humans. It is well known that Mycobacterium bovis and other species in the M. tuberculosis complex (MTC) can cause respiratory diseases as zoonosis. We analyzed the MTC isolates collected from tuberculosis patients from Japan in 2002 using a multiplex PCR system that detected cfp32, RD9 and RD12. A total of 970 MTC isolates that were representative of the tuberculosis cases throughout Japan, were examined using this method. As a result, 966 (99.6%) M. tuberculosis, two Mycobacterium africanum and two Mycobacterium canettii were identified using a multiplex PCR system, while no M. bovis was detected. Two isolates that lacked RD9 were initially considered to be M. canettii, but further analysis of the hsp65 sequence revealed them to be M. tuberculosis. Also two M. africanum were identified as M. tuberculosis using the −215 narG nucleotide polymorphism. Though PCR-linked methods have been used for a rapid differentiation of MTC and NTM, from our cases we suggest careful interpretation of RD based identification. [Copyright &y& Elsevier]

Summary: Many nontuberculous mycobacteria (NTM) species have clinical significance, and the rapid and reliable identification of Mycobacterium tuberculosis complex (MTBC) and NTM species is important. We evaluated the simultaneous detection of MTBC and NTM in respiratory specimens. MTBC and NTM were simultaneously detected and identified by laboratory-developed (LDT) real-time PCR, multiplex real-time PCR/melting curve analysis, rpoB PCR restriction fragment length polymorphisms and the AdvanSure Mycobacteria GenoBlot assay (LG Life Sciences). Eighty-five respiratory specimens from 69 patients showed simultaneous detection of MTBC and NTM. A line probe assay showed 70.6% concordance with LDT. Ten patients (14.5%) had a history of tuberculosis, and eight patients (11.6%) had been previously diagnosed with bronchiectasis. Mixed cultures were present one time in 57 patients (82.6%) and repeatedly in 12 patients (17.4%). MTBC was more frequent in 44 patients (63.8%), and NTM was isolated in seven patients (10.1%). The commonly detected NTM species in the mixed cultures were Mycobacterium intracellulare (29.0%) and Mycobacterium abscessus (29.0%). Co-isolation caused a failure of antitubercular drug susceptibility testing in 2 patients (2.9%). Molecular methods allow MTBC and NTM species to be simultaneously identified in respiratory specimens. NTM isolated with MTBC has clinical significance in some patients and should not be ignored. [Copyright &y& Elsevier]

Summary: Objective: We did a systematic review and meta-analysis of published studies to evaluate the accuracy of commercial MPT64-based immunochromatographic tests for rapid identification of Mycobacterium tuberculosis complex. Methods: We identified studies by searching Pubmed, BIOSIS Previews and Web of Science, and included studies using predetermined inclusion criteria. The data were pooled using the DerSimonian-Laird random effects model. Results: A total of 28 studies were included in the final analysis. Pooled estimates were 97% (confidence interval [CI] 96–97%) for sensitivity and 98% (CI 98–99%) for specificity. The summary receiver operating characteristic curve showed an area of 0.9968 and a Q* of 0.98. Subgroup analysis showed that test accuracy did not depend on commercial kit, reference test and medium. Conclusions: Commercial MPT64-based immunochromatographic tests are highly sensitive and specific for rapid identification of M. tuberculosis complex. They are good alternatives to biochemical test and molecular assays. Nevertheless, additional studies are required in setting with high prevalence of mpt64 mutations or high contamination of cultures. [Copyright &y& Elsevier]

Summary: The mycobacterial antigen PstS1 is a highly immunogenic and immunostimulatory component of the mycobacterial cell membrane and a good candidate for the diagnosis and vaccination against tuberculosis. Here we selected 180 clinical isolates of Mycobacterium tuberculosis complex (MTBC) in China and 11 different Bacille Calmette Guerin (BCG) strains, amplified the gene of the PstS1 antigen and compared the sequences with those of four other Mycobacterium bovis and BCG strains from the NCBI genome website. Some of the mutations, especially 2 frameshift mutations, occurred in the PstS1antigen, which may have resulted in the protein function alteration and ongoing immune evasion. A unique single nucleotide polymorphism of the M. bovis and BCG strains was found in this antigen and may be useful for differentiating M. bovis and BCG strains from M. tuberculosis strains. [Copyright &y& Elsevier]

Highlights: [•] We characterized MTBC populations from Portugal and Northeast Brazil. [•] We used a SNP-based Bayesian tree for identification of the MTBC isolates. [•] Our approach allowed to define the usefulness of SNPs in strain identification. [•] MTBC populations from Portugal and Northeast Brazil presented striking similarities. [ABSTRACT FROM AUTHOR]

Summary: Objectives: A fundamental to global tuberculosis (TB) control is timely and accurate diagnosis of infectious cases of the disease. Among various methods, techniques based on nucleic acid amplification are the ones with promising prospects. The present study evaluates the diagnostic value of the recently developed IS6110-based loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis complex (MTBC) in sputum specimens. Methods: In this cross-sectional study (2008–2009), IS6110-LAMP was evaluated on 101 sputum specimens from 93 highly suspected TB patients and compared to Amplicor MTB test and in-house IS6110-PCR and -nested PCR assays. Culture results or clinical recovery following anti-TB therapy was considered as a reference to prove the TB cases. Results: The overall sensitivity of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 89.6% (69/77 specimens; 95% confidence interval [CI], 80.5–95.4%), 76.6% (59/77 specimens; CI, 65.6–85.5%), 79.2% (61/77 specimens; CI, 68.5–87.6%) and 59.7% (46/77 specimens; CI, 47.9–70.8%). The specificity and positive predictive value (PPV) were 100% for all the tests, and the negative predictive value (NPV) of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 75%, 57.1%, 60%, and 43.6%. There was an excellent overall agreement between LAMP and nPCR (k 0.828), and between LAMP and Amplicor (k 0.746), in addition to a better tolerance of IS6110-LAMP to inhibitors present in clinical specimens. Conclusion: The better diagnostic performance of IS6110-LAMP compared to Amplicor (p = 0.009), nPCR (p = 0.013) and PCR (p < 0.0001) besides its rapidity, simplicity, and cost-effectiveness makes it a valuable method for the detection of MTBC in clinical samples, particularly in resource-limited settings. [Copyright &y& Elsevier]

Abstract: Objectives: This work aims to develop rapid nano-gold assay prototypes for specific detection of Mycobacterium tuberculosis complex (MTBC). Design and methods: Spherical gold nanoparticles (AuNPs, 14nm) were synthesized by citrate reduction method and characterized by spectrophotometry and SEM. MTB 16s rDNA regions were amplified by PCR and amplicons were detected using genus- and species-specific oligotargeters and AuNPs. In a second prototype, MTBC unamplified genomic DNA was directly detected using species-specific oligo-targeters and AuNPs. Results: Detection limits were 1ng for PCR product and 40ng for genomic DNA. The nano-gold prototype detected 45 positive genomic DNA samples which were also positive with automated liquid culture system (BACTEC™ MGIT™) and semi-nested PCR (100% concordance). Following DNA extraction, using standard procedures, the TB nano-gold prototype turnaround time is about 1h. Conclusions: We have developed nano-gold assay prototype for direct and inexpensive detection of MTBC. The developed prototypes are simple, sensitive, rapid and can substitute PCR-based detection. The developed assay may show potential in the clinical diagnosis of TB especially in developing countries. [Copyright &y& Elsevier]

Abstract: Molecular identification of mycobacteria in positive Mycobacteria Growth Indicator Tube (MGIT) cultures can accelerate mycobacterial diagnosis. A membrane hybridization array (Blue Point) was evaluated for this purpose in 284 positive MGIT cultures. Discrepant results were resolved by testing with the GenoType Mycobacterium kit, TBc ID test, sequencing of the 16S rRNA gene and internal transcribed spacer. Total recovery from culture and the array (if confirmed) was considered 100%. The sensitivity, specificity, positive, and negative predictive values of the array for detection of Mycobacterium tuberculosis complex were 99.4%, 100%, 100%, and 99.2%, respectively, while the corresponding values of culture were 95.1%, 100%, 100%, and 93.8%, respectively, with significant differences in sensitivity and negative predictive value being found between the 2 methods. The recoveries of nontuberculous mycobacteria and mixed cultures of the array were also significantly higher than those of culture. The array can be adopted in routine mycobacteriology laboratory. [Copyright &y& Elsevier]

Summary: The real-time PCR with duplex primer sets and the MPB64-based immunochromatographic assay are newly developed methods for rapid differentiation of mycobacteria. The aim of this study is to evaluate the two methods for differentiation between Mycobacterium tuberculosis complex and nontuberculous mycobacteria. A total of 95 clinical mycobacterial isolates belonging to 22 different species and 16 reference strains of 16 different species were differentiated by duplex real-time PCR method and MPB64-based immunochromatographic assay method. The two methods were evaluated by comparison with conventional biochemical technique as the gold standard method. The duplex real-time PCR method correctly differentiated all reference strains as well as the MPB64-based immunochromatographic assay method. For clinical isolates, the accuracy of the duplex real-time PCR method (100%) was slightly higher than the MPB64-based immunochromatographic assay method (97.9%), but there was no statistical significance between the two methods (P > 0.05), and there was an excellent agreement between them (Kappa = 0.957). The duplex real-time PCR method possesses greater potential for differentiation of mycobacteria in the clinical laboratory than the MPB64-based immunochromatographic assay method. However, the MPB64-based immunochromatographic assay method is more convenient than the duplex real-time PCR method when the number of sample is small. [ABSTRACT FROM AUTHOR]

Abstract: Clinical reports for Eleonora of Toledo (1522–1562), the wife of Cosimo I de’ Medici, imply that during her 28th year she developed pulmonary tuberculosis, which was complicated by an attack of pernicious malaria, killing her at age 40. Eleonora''s autopsy indicated that she had severe lung lesions consistent with chronic pulmonary infection. To clarify her disease status, we performed paleomolecular investigations. Our results identified ancient DNA from the Mycobacterium tuberculosis complex (MTB), along with Leishmania infantum (VL). Our data are of particular interest since in Tuscany the endemic foci of L. infantum are widely distributed and overlapped with those of malaria prior to its eradication. Although we can only speculate about Eleonora''s true state of health, this clear evidence of long-term co-infection with MTB and VL is of major medical and biological interest since the co-evolution of the two pathogens and host–pathogen interactions in co-infected individuals are still not fully understood. [Copyright &y& Elsevier]

Abstract: Molecular diagnostics and genotyping of pathogens have become indispensable tools in clinical microbiology and disease surveillance. For isolates of the Mycobacterium tuberculosis complex (MTBC, causative agents of tuberculosis), multilocus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible, and discriminatory typing method. Here, we review new sets of specialized web based bioinformatics tools that have become available for analyzing MLVA data especially in combination with other, complementary genotyping markers (polyphasic analysis). Currently, there are only two databases available that are not restricted to store one kind of genotyping data only, namely SITVIT/SpolDB4 and MIRU-VNTRplus. SITVIT/SpolDB4 (http://www.pasteur-guadeloupe.fr:8081/SITVITDemo) contains spoligotyping data from a large number of strains of diverse origin. However, besides options to query the data, the actual version of SITVIT/SpolDB4 offers no functionality for more complex analysis e.g. tree-based analysis. In comparison, the MIRU-VNTRplus web application (http://www.miru-vntrplus.org), represents a freely accessible service that enables users to analyze genotyping data of their strains alone or in comparison with a currently limited but well characterized reference database of strains representing the major MTBC lineages. Data (MLVA-, spoligotype-, large sequence polymorphism, and single nucleotide polymorphism) can be visualized and analyzed using just one genotyping method or a weighted combination of several markers. A variety of analysis tools are available such as creation of phylogenetic and minimum spanning trees, semi-automated phylogenetic lineage identification based on comparison with the reference database and mapping of geographic information. To facilitate scientific communication, a universal, expanding genotype nomenclature (MLVA MtbC15-9 type) service that can be queried via a web- or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions. Perspectives for future development, including generalization to other bacterial species, are presented. [Copyright &y& Elsevier]

Abstract: Paleogenetic analysis for tuberculosis (TB) was conducted on bone and sediment samples dating from the 17th to 19th centuries from the archeological site of Nossa Senhora do Carmo Church in Rio de Janeiro, Brazil. Forty samples were analyzed, corresponding to 32 individuals from 28 burials, 22 of primary type and 6 of secondary type. The samples were collected following strict paleogenetic investigation guidelines and submitted to ancient DNA (aDNA) extraction. In order to detect TB infection, aDNA hybridizations with the molecular targets of Mycobacterium tuberculosis complex (MTC) IS6110 and IS1081 were applied. Additionally, the ancestry of individuals was assessed by human mitochondrial DNA (mtDNA) analysis of hypervariable segment I (HVS-I) sequence polymorphisms. The results of aDNA hybridizations demonstrated varying levels of MTC intensity in 17/32 individuals (53.1%), using the IS6110 target. The IS1081 MTC target showed lower sensitivity, confirming TB positivity in 10/32 (31.2%) individuals. The mtDNA analysis allowed the recovery of HVS-I sequences in 23/32 individuals (71.8%). The majority of these individuals (21/23, 91.3%) were of European ancestry, especially in primary burials. Haplogroups U, J, V, T, K, N, H and R, were identified with haplogroup U being the most frequent at 6/23 (26.1%). African and Amerindian mtDNA haplogroups were observed in two individuals in secondary burials. In spite of the ecclesiastic and aristocratic bias of the population of the study, human ancestry analysis revealed the prominent contribution of Europeans in the introduction or spread of TB in the New World. [Copyright &y& Elsevier]

Abstract: The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchi''s protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S–23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance. [Copyright &y& Elsevier]

Summary: Mycobacterium microti is a member of the Mycobacterium tuberculosis complex (MTC). M. microti is generally considered a pathogen of small rodents, although sporadic infections in a range of other mammals, including domestic animals and man, have been reported. While many human infections have been associated with immunosuppression, an increasing number of cases are being reported in immunocompetent patients. Two cases of M. microti infection in meerkats (Suricata suricatta) are reported. These are the first cases of mycobacterial disease to be described in meerkats outside Africa. [ABSTRACT FROM AUTHOR]

Abstract: From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1–18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19–48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans. [Copyright &y& Elsevier]

Abstract: Tuberculosis remains a major public health problem in Nigeria. While human to human transmission of Mycobacterium tuberculosis is clearly of major importance in driving the tuberculosis epidemic in Nigeria, the impact of Mycobacterium bovis transmission from infected cattle is largely unknown. Molecular epidemiology of M. bovis in Nigeria will increase our understanding of this endemic disease and provide tools to assess cattle-to-human transmission. Between 2002 and 2004, molecular techniques including spoligotyping, variable number of tandem repeats (VNTR) typing and deletion typing were used to track and analyze a sample of strains of the M. tuberculosis complex circulating in the cattle population in Ibadan, Southwestern Nigeria. In all, 180 isolates were typed with a view to elucidating epidemiological information on circulating strains, occurrence of transborder transmission and molecular diversity of the M. bovis strains. Results obtained showed that 99% (178/180) of the isolates were M. bovis, while the remaining were M. tuberculosis and M. africanum. In all, strains of M. bovis had 34 different spoligotypes: strains with spoligotype pattern SB0944 (as designated by www.mbovis.org) were the most common (46% of strains). This molecular type is also common in countries neighbouring Nigeria. Strains with this spoligotype pattern could be further divided into 40 different VNTR types. This analysis shows the value of simple molecular epidemiological techniques applied to strains of M. bovis and suggests that further epidemiological studies will shed more light on the transmission dynamics of bovine tuberculosis locally and across neighbouring African countries. [Copyright &y& Elsevier]