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We identified tuberculosis in 1,836 macaques from 6 wild rhesus (Macaca mulatta), 23 common long-tailed (M. fascicularis fascicularis), and 6 Burmese long-tailed (M. fascicularis aurea) macaque populations in Thailand. We captured, anesthetized, and collected throat, buccal, and rectal swab specimens from the macaques. We screened swabs for Mycobacterium tuberculosis complex (MTBC) using insertion sequence 6110-specific nested PCR. We found higher MTBC prevalence at both population and individual levels among M. mulatta than M. fascicularis fascicularis macaques; all 3 M. fascicularis aurea macaque populations were positive for tuberculosis. We found that throat swab specimens provided the best sample medium for detecting MTBC. Our results showed no difference in MTBC prevalence between male and female animals, but a higher percentage of adults were infected than subadults and juveniles. Although we detected no association between frequency of human-macaque interaction and MTBC prevalence, bidirectional zoonotic transmission should be considered a possible public health concern.

This study presents a novel multiplex assay based on capillary electrophoresis (CE) for the simultaneous detection of three Mycobacterium tuberculosis complex (MTBC) genes: IS6110 , rpoB , and HSP65 . Unlike conventional molecular diagnostic methods that target only a single gene, which may lead to misdiagnosis or missed diagnosis, this CE-based multiplex approach provides comprehensive detection to reduce diagnostic errors. Specificity testing with 76 microorganisms representing common respiratory pathogens confirmed 100% analytical specificity with no cross-reactivity, while sensitivity analysis demonstrated detection limits ranging from 10 to 20 copies/mL for all three target genes. In a prospective clinical validation study of 1067 patients suspected of pulmonary tuberculosis, the multiplex assay showed 77.4% sensitivity (CI 74.9%–79.9%), 99.6% specificity (CI 99.2%–100%), 96.0% positive predictive value (CI 94.8%–97.2%), and 97.1% negative predictive value (CI 96.1%–98.1%). Notably, the study identified 6 MTBC strains (4.8% of TB patients) with IS6110 deletions through whole-genome sequencing, which would result in false-negative results for any commercial PCR kits targeting IS6110 . This integrated multiplex approach enhances diagnostic accuracy by simultaneously targeting multiple genes; then it offers the potential to reduce misdiagnosis and missed diagnosis of tuberculosis. In summary, the multiplex assay provides a more comprehensive alternative to current single-target molecular methods for MTBC detection. Key points • The multiplex assay provides one-run results for IS6110, rpoB, and HSP65. •  The multiplex assay is a more comprehensive method to detect MTBC. •  This approach can reduce misdiagnosis and missed diagnosis of TB.

Background Tuberculosis is still a significant diagnostic and therapeutic challenge with high proportion of smear- and culture- negative incidences worldwide. The conventional diagnostic tests are time-consuming and have a low sensitivity. Digital PCR is a novel technology which can detect target sequences with relatively low abundance and obtain the absolute copy numbers of the targets. Methods We assessed the accuracy of dPCR in TB diagnosis using more than 250 specimens, and for the first time, we selected M.tuberculosis -specific IS1081 in addition to widely used IS6110 as the amplification targets for dPCR. The quantification of target DNA was calculated using QuantaSoft Version 1.7.4.0917 (BioRad), and SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. Results IS6110-dPCR was more sensitive than IS1081, with the sensitivity and specificity accounting for 40.6 and 93.4% respectively. When we classified the TB patients by personal factors for high copy number of M.tuberculosis derived DNA in plasma: bilateral TB, extrapulmonary TB and disseminated TB, the sensitivity of both IS6110- and IS1081- dPCR was the highest in patients with disseminated TB (IS6110, 100%; IS1081, 68.8%), while their sensitivity was a bit higher in patients with extrapulmonary TB (IS6110, 50.0%; IS1081, 39.3%) than that in bilateral TB (IS6110, 43.3%; IS1081 , 33.3%). Compared with traditional TB diagnostic tests, joint detection IS6110 & IS1081-dPCR was not as sensitive as smear microscope or mycobacterial culture, but it was higher than IS6110 qPCR ( p  < 0.05) and was able to detect 47.4% of smear-negative TB patients. Conclusion Our study suggested that plasma IS6110-dPCR is a rapid, moderate accurate and less-invasive method to detect M.tuberculosis DNA in plasma of TB patients and IS6110 & IS1081-dPCR has a potential to aid diagnosis of smear-negative TB.

Background Detecting tuberculosis (TB) remains challenging in low‐resource settings due to limited access to advanced diagnostic tools. The emergence of isothermal amplification techniques, such as loop‐mediated isothermal amplification (LAMP), presents a promising solution for TB detection. Several isothermal amplification techniques, e.g., strand displacement amplification (SDA), helicase‐dependent amplification (HDA), recombinase polymerase amplification (RPA), and rolling cycle amplification (RCA), have been attracting attention for development as point‐of‐care testing (POCT) in many diagnostic fields. The present review summarizes the most common isothermal amplification techniques for the detection of TB. Methods To collect the necessary information, we searched PubMed and Scopus for scientific evidence published from 2000 to September 2024, using the keywords loop‐mediated isothermal amplification tuberculosis, strand displacement amplification tuberculosis, helicase‐dependent amplification tuberculosis, recombinase polymerase amplification tuberculosis, rolling circle amplification tuberculosis, polymerase spiral reaction tuberculosis, cross‐priming amplification tuberculosis, and multiple cross displacement amplification tuberculosis. Results The methodologies of the most usual isothermal amplification techniques are addressed, as well as the advantages and limitations of the technique, highlighting applications in TB diagnosis. Exploring these advances addresses the principles of innovation in isothermal amplification, including their performance and applicability across diverse sample types. Some techniques have already been launched into routine TB diagnosis, while other promising tests are still being researched or evaluated. These techniques are easy to perform and provide results rapidly, enabling prompt treatment within a few hours. Isothermal amplification techniques may be one of the key molecular tools to fight TB in pursuit of the goals of the End TB strategy. The emergence of isothermal amplification techniques presents a promising solution for tuberculosis (TB) diagnosis. These methods are attractive due to their simplicity, speed, and ability to perform without specialised equipment, which could make them suitable for low‐resource settings. Therefore, isothermal amplification techniques may be one of the key molecular tools used to fight TB in pursuit of the goals of the End TB strategy.

Mycobacterium orygis, a subspecies of the Mycobacterium tuberculosis complex (MTBC), has emerged as a significant concern in the context of One Health, with implications for zoonosis or zooanthroponosis or both. MTBC strains are characterized by the unique insertion element IS6110, which is widely used as a diagnostic marker. IS6110 transposition drives genetic modifications in MTBC, imparting genome plasticity and profound biological consequences. While IS6110 insertions are customarily found in the MTBC genomes, the evolutionary trajectory of strains seems to correlate with the number of IS6110 copies, indicating enhanced adaptability with increasing copy numbers. Here, we present a comprehensive analysis of IS6110 insertions in the M. orygis genome, utilizing ISMapper, and elucidate their genetic consequences in promoting successful host adaptation. Our study encompasses a panel of 67 paired-end reads, comprising 11 isolates from our laboratory and 56 sequences downloaded from public databases. Among these sequences, 91% exhibited high-copy, 4.5% low-copy, and 4.5% lacked IS6110 insertions. We identified 255 insertion loci, including 141 intragenic and 114 intergenic insertions. Most of these loci were either unique or shared among a limited number of isolates, potentially influencing strain behavior. Furthermore, we conducted gene ontology and pathway analysis, using eggNOG-mapper 5.0, on the protein sequences disrupted by IS6110 insertions, revealing 63 genes involved in diverse functions of Gene Ontology and 45 genes participating in various KEGG pathways. Our findings offer novel insights into IS6110 insertions, their preferential insertion regions, and their impact on metabolic processes and pathways, providing valuable knowledge on the genetic changes underpinning IS6110 transposition in M. orygis.

Bovine tuberculosis (bTB) caused by complex (MTC) remains a significant concern for public health. Direct real-time PCR and droplet digital PCR (ddPCR) are proposed as alternative tools to enhance diagnostic precision and efficiency. This study aims to assess the diagnostic performance of a ddPCR assay targeting IS for the detection of MTC DNA in both microbiological culture and fresh lymph node (LN) tissue samples obtained from cattle, in comparison with the established reference standard, the microbiological culture followed by real-time PCR. The fresh LNs (N=100) were collected each from a different cattle carcass at the slaughterhouse. The limit of detection of ddPCR-IS was set to 101 copies per 20 μl reaction. DdPCR-IS detected 44 out of 49 reference-standard positive samples and yielded negative results in 47 out of 51 reference-standard negative samples, resulting in adjusted sensitivity (Se) and specificity (Sp) of 90.76% [95% confidence interval (CI): 82.58 - 98.96%)], and 100% (95% CI: 100%) respectively. The estimated adjusted false negative rate (FNR) was 9.23% (95% CI: 1.04 - 17.42%) and the false positive rate (FPR) was 0% (95% CI: 0%). When directly applied from fresh bovine LN tissues, ddPCR-IS6110 identified 47 out of 49 reference-standard positive samples as ddPCR-IS6110-positive and 42 out of 51 reference-standard negative samples as ddPCR-IS -negative, resulting in adjusted Se and Sp values of 94.80% [95% (CI): 88.52 - 100%] and 100% (95% CI: 100%), respectively. The adjusted FNR was 5.20% (95% CI: 0 - 11.50%) and the FPR was 0% (95% CI: 0%). Noteworthy, ddPCR-IS disclosed as positive 9 samples negative to reference-standard. DdPCR-IS proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method.

Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis , the leading cause of bacterial infection worldwide. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including Mycobacterium tuberculosis , the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in M. tuberculosis strain genotyping, its evolutionary path in Mycobacteriaceae is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in M. tuberculosis complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and Mycobacterium canettii from a Streptococcus -like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in M. tuberculosis lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS 6110 . The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS 6110 insertion. We show in M. tuberculosis H37Rv that the repeat at the 5′ end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in M. canettii . This study retracing the evolutionary events of HGT and IS 6110 -driven genomic deletions helps us to better understand the strain-specific variations in M. tuberculosis lineages. IMPORTANCE Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis , the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in M. tuberculosis has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and M. canettii , by HGT-mediated events. The most likely source of HGT was an environmental Firmicutes bacterium. Genomic mapping of the CRISPR loci showed the IS 6110 transposition-driven variations in M. tuberculosis strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in M. tuberculosis lineages.

This study aimed to evaluate the accuracy of detecting drug-resistant complex (MTBC)-specific DNA in sputum specimens from 48 patients diagnosed with pulmonary tuberculosis. The presence of MTBC DNA in the specimens was validated using the GeneXpert MTB/RIF system and compared with a specific PCR assay targeting the IS and the 40 gene sequence fragments. Additionally, the results obtained by multiplex PCR assays to detect the most frequently encountered rifampin, isoniazid, and ethambutol resistance-conferring mutations were matched with those obtained by GeneXpert and phenotypic culture-based drug susceptibility tests. Of the 48 sputum samples, 25 were positive for MTBC using the GeneXpert MTB/RIF test. Nevertheless, the IS and 40 single-step PCR revealed the IS in 27 of the 48 sputum samples, while the 40 gene fragment was found in only 17 of them. Furthermore, multiplex PCR assays detected drug-resistant conferring mutations in 21 (77.8%) of the 27 samples with confirmed MTBC DNA, 10 of which contained single drug-resistant conferring mutations towards ethambutol and two towards rifampin, and the remaining nine contained double-resistant mutations for ethambutol and rifampin. In contrast, only five sputum specimens (18.5%) contained drug-resistant MTBC isolates, and two contained mono-drug-resistant MTBC species toward ethambutol and rifampin, respectively, and the remaining three were designated as multi-drug resistant toward both drugs using GeneXpert and phenotypic culture-based drug susceptibility tests. Such discrepancies in the results emphasize the need to develop novel molecular tests that associate with phenotypic non-DNA-based assays to improve the detection of drug-resistant isolates in clinical specimens in future studies.

Background Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease. Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries. Methods In the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process. To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB. Results Our SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA. Conclusion To our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.

Background CRISPR and CRISPR-flanking genomic regions are important for molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) strains, and potentially for adaptive immunity to phage and plasmid DNA, and endogenous roles in the bacterium. Genotyping in the Israel National Mycobacterium Reference Center Tel-Aviv of over 1500 MTBC strains from 2008–2013 showed three strains with validated negative 43-spacer spoligotypes, that is, with putatively deleted direct repeat regions (deleted-DR/CRISPR regions). Two isolates of each of three negative spoligotype MTBC (a total of 6 isolates) were subjected to Next Generation Sequencing (NGS). As positive controls, NGS was performed for three intact-DR isolates belonging to T3_Eth, the largest multiple-drug-resistant (MDR)-containing African-origin cluster in Israel. Other controls consisted of NGS reads and complete whole genome sequences from GenBank for 20 intact-DR MTBC and for 1 deleted-DR MTBC strain recognized as CAS by its defining RD deletion. Results NGS reads from negative spoligotype MTBC mapped to reference H37Rv NC_000962.3 suggested that the DR/CRISPR regions were completely deleted except for retention of the middle IS6110 mobile element. Clonally specific deletion of CRISPR-flanking genes also was observed, including deletion of at least cas2 and cas1 genes. Genomic RD deletions defined lineages corresponding to the major spoligotype families Beijing, EAI, and Haarlem, consistent with 24 loci MIRU-VNTR profiles. Analysis of NGS reads, and analysis of contigs obtained by manual PCR confirmed that all 43 gold standard DR/CRISPR spacers were missing in the deleted-DR genomes. Conclusions Although many negative spoligotype strains are recorded as spoligotype-international-type (SIT) 2669 in the SITVIT international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least 4 different 24 loci MIRU-VNTR and RD deletion families. We report for the first time negative spoligotype-associated total loss of CRISPR region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least CRISPR-associated genes cas2 and cas1 . Since cas1 deleted E.coli shows increased sensitivity to DNA damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-DR strains and Beijing family strains as both lack the cas1 gene.