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Background: The growing use of real-time PCR (qPCR) as a diagnostic method for bovine TB (bTB) requires rapid and effective DNA extraction methods, which are crucial for its success. Automated DNA extraction methods based on magnetic beads are a promising alternative to conventional silica column-based protocols (COL protocol) due to their high throughput capacity and reduced hands-on time. This study aimed to assess the performance of the MagMax CORE Nucleic Acid Purification kit and the KingFisher Flex instrument (KF protocol) as an alternative for scaling up the use of qPCR in bTB diagnosis. Methodology: Performance was evaluated with two different real-time PCR (qPCR) protocols, based on the IS6110 element and the QuantiFast and VetMAX™ (QF and VM protocols) kits, on 145 frozen tissue homogenates confirmed as either bTB-positive or negative through a composite reference standard based on microbiological culture, column-based extraction, and qPCR, as well as on negative tissue samples spiked with 106 to 103 CFU/ml of M. bovis BCG. Results: The performance of both qPCR protocols was very high on samples extracted using the KF protocol, with positive percent agreement (PPA) values of 89.04% [95% Confidence Interval (CI): 79.54–95.15%] and 93.15% [95% CI: 84.74–97.74%] for the QF and VM protocols, respectively, and negative percent agreement (NPA) values of 100% [95% CI: 95.01–100.00%]. A higher variability was identified in samples analysed with the same qPCR protocol but different extraction methods. Higher Ct values were identified for samples extracted using the KF protocol in both routine and spiked samples, likely due to using the same amount of starting material for both extraction methods, which was lower than recommended by the manufacturer for the KF protocol. Discussion: The results of this study indicate that the MagMAX CORE Nucleic Acid Purification kit coupled with a KingFisher Flex instrument is a valuable alternative for the extraction of MTBC DNA from bovine tissues. However, the increased variability and Ct values suggest that a larger amount of starting material is recommended for this methodology, warranting further studies. [ABSTRACT FROM AUTHOR]

The franciscana (Pontoporia blainvillei) is a small cetacean endemic to the southwestern Atlantic Ocean in a vulnerable conservation status. Their habitat is restricted to shallow waters and estuaries. In 2020, fresh female carcass of franciscana (juvenile/subadult) was found dead alone (individual stranding) on a public access beach in the Rio Negro Estuary in "Balneario El Cóndor", Argentinian Patagonia. Grossly, a widespread granulomatous lesion compatible with tuberculosis were observed. A sample of mesenteric lymph nodes with granulomatous lesions (~ 50 g) was collected for bacteriological culture and molecular identification. Bacterial growth was observed in Stonebrink media and Ziehl Neelsen staining revealed acid-fast bacilli. Identification of a Mycobacterium bovis strain with the SB0288 spoligotype was obtained. The spoligotype detected here has already been reported in cattle and humans. Presumably, fluids and feces of infected livestock and wild animals, or their carcasses, may contaminate the water. Thus, this report demonstrates the potential risk of zoonotic tuberculosis transmission through wastewater. Contaminated wastewater is eventually a threat to animal species living in the area, and potentially becomes a zoonotic risk. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays. Results: The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria. Conclusions: In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays. Results The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria. Conclusions In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment.

Abstract Background Bovine tuberculosis (bTB) is a chronic disease caused by members of the Mycobacterium tuberculosis complex (MTBC) that ultimately leads to the development of progressive granulomatous lesions. Although the disease is widespread, especially in crossbred cattle in Ethiopia, routine investigations and surveillance are lacking. Thus, the aim of this study was to determine the prevalence, associated risk factors, and species of mycobacteria causing bTB in slaughtered cattle at four slaughterhouses in Central Ethiopia. Methods Postmortem examination of 7,640 cattle was conducted using a cross-sectional slaughterhouse survey. A total of 388 tuberculous-like lesions (TBLs) were collected from 173 animals and cultured. Six target genes were used to differentiate mycobacterial species using multiplex real-time PCR (mRT-PCR). Multivariate logistic regression analyses and related odds ratios (ORs) were used to gauge the strength of the associations between risk factors, TBL incidence and culture growth. Results The prevalence of TBL was 2.3% (95% CI = 2.0-2.6). Logistic regression analysis indicated an increased risk of TBL in crossbred cattle (OR = 11.8, 95% CI: 6.4, 21.2, p

Abstract Background The respiratory chain plays a key role in the growth of Mycobacterium tuberculosis complex (MTBC). However, the exact regulatory mechanisms of this system still need to be elucidated, and only a few studies have investigated the impact of genetic mutations within the respiratory chain on MTBC transmission. This study aims to explore the impact of respiratory chain gene mutations on the global spread of MTBC. Results A total of 13,402 isolates of MTBC were included in this study. The majority of the isolates (n = 6,382, 47.62%) belonged to lineage 4, followed by lineage 2 (n = 5,123, 38.23%). Our findings revealed significant associations between Single Nucleotide Polymorphisms (SNPs) of specific genes and transmission clusters. These SNPs include Rv0087 (hycE, G178T), Rv1307 (atpH, C650T), Rv2195 (qcrA, G181C), Rv2196 (qcrB, G1250T), Rv3145 (nuoA, C35T), Rv3149 (nuoE, G121C), Rv3150 (nuoF, G700A), Rv3151 (nuoG, A1810G), Rv3152 (nuoH, G493A), and Rv3157 (nuoM, A1243G). Furthermore, our results showed that the SNPs of atpH C73G, atpA G271C, qcrA G181C, nuoJ G115A, nuoM G772A, and nuoN G1084T were positively correlated with cross-country transmission clades and cross-regional transmission clades. Conclusions Our study uncovered an association between mutations in respiratory chain genes and the transmission of MTBC. This important finding provides new insights for future research and will help to further explore new mechanisms of MTBC pathogenicity. By uncovering this association, we gain a more complete understanding of the processes by which MTBC increases virulence and spread, providing potential targets and strategies for preventing and treating tuberculosis.

Background: Bovine tuberculosis (bTB) is a chronic disease caused by members of the Mycobacterium tuberculosis complex (MTBC) that ultimately leads to the development of progressive granulomatous lesions. Although the disease is widespread, especially in crossbred cattle in Ethiopia, routine investigations and surveillance are lacking. Thus, the aim of this study was to determine the prevalence, associated risk factors, and species of mycobacteria causing bTB in slaughtered cattle at four slaughterhouses in Central Ethiopia. Methods: Postmortem examination of 7,640 cattle was conducted using a cross-sectional slaughterhouse survey. A total of 388 tuberculous-like lesions (TBLs) were collected from 173 animals and cultured. Six target genes were used to differentiate mycobacterial species using multiplex real-time PCR (mRT-PCR). Multivariate logistic regression analyses and related odds ratios (ORs) were used to gauge the strength of the associations between risk factors, TBL incidence and culture growth. Results: The prevalence of TBL was 2.3% (95% CI = 2.0-2.6). Logistic regression analysis indicated an increased risk of TBL in crossbred cattle (OR = 11.8, 95% CI: 6.4, 21.2, p < 0.001). Animals slaughtered at Adama (OR = 3.2, 95% CI: 1.2, 7.3, p = 0.009) or Burayu (OR = 5.8, 95% CI: 3.9, 8.9, p < 0.001) had a greater risk of TBL than those slaughtered at Sululta. There were significantly more TBL-positive lesions in the lungs and lymph nodes related to the lung (OR = 7.1; 95% CI: 2.7, 24.5, p < 0.001) and the head lymph node (OR = 5.6; 95% CI: 1.8, 21.7; p = 0.006) compared to gut associated lymph nodes. Among the 173 TBL-positive animals, 36% (95% CI = 28.8, 43.2), and among the 388 TBL-positive tissues, 24.2% (95% CI = 20, 29) were culture and mRT-PCR positive. All the culture-generated isolates were positive for M. bovis in mRT-PCR. Among them, two animals had mixed infections including one zebu cattle tested positive for both M. caprae and M. bovis, and a crossbred cow tested positive for both M. tuberculosis and M. bovis in mRT-PCR. This suggests persistent transmission within the cattle population, posing a substantial public health threat. Conclusion: This study revealed an eleven-fold greater risk of bTB-related lesions in crossbred cattle compared to local zebu cattle. This finding highlights the necessity for targeted interventions, continuous vigilance, and thorough carcass inspection to mitigate public health risks. [ABSTRACT FROM AUTHOR]

Animal tuberculosis (TB) is often maintained by multi-host communities, including livestock and wildlife. Quantitative studies of such communities require estimating the true prevalence of TB, correcting the apparent prevalence by the diagnostic sensitivity (Se) and specificity (Sp) of the test. The goal of this study was to lay the foundations for estimating the true prevalence of TB in wild ungulate populations (wild boar and two cervids: red deer and fallow deer). We used Bayesian latent class models to assess the Se and Sp of gross pathology, IS6110 real-time PCR in tissues, bacteriological culture, and P22 indirect ELISA. We analyzed 308 harvested wild ungulates (211 wild boar and 97 cervids: 92 red deer and 5 fallow deer). The Se of bacteriological culture (80.4%, CI95 61.0-96.3%) and gross pathology (87.9%, CI95 69.5-99.9%) was reasonably good in wild boar. These tests showed lower Se in cervids: 60.2% (CI95 38.3-82.3%) for bacteriological culture and 81.5% (CI95 63.6-96.2%) for gross pathology. The Se of the real-time PCR was low (50.7% in wild boar and 53.0% in cervids). These tests showed Sp between 95.2 and 99.1% in both taxa. The P22 ELISA performed reasonably well in wild boar (Se = 71.9%, CI95 59.2-83.4%; Sp = 98.8%, CI95 96.9-99.9%) but lacked Sp in cervids (Se = 77.1%, CI95 62.9-89.7%; Sp = 74.5%, CI95 65.7-83.3%). The real-time PCR in wild boar and cervids and bacteriological culture in cervids tended to show higher Se in low-prevalence populations, possibly due to a higher proportion of early-stage TB lesions. In cervids, the parallel interpretation of gross pathology and bacteriological culture significantly improved the diagnostic performance (Se = 93.1%, CI95 84.7-98.9%; Sp = 92.9%, CI95 86.0-98.3%). Our results allow the estimation of true prevalence from the results of a single diagnostic test applied to harvested wild boar, red deer, and fallow deer, paving the way for more precise quantitative ecological studies of the multi-host TB maintenance community. [ABSTRACT FROM AUTHOR]

Background: Pulmonary tuberculosis (TB) is predominantly caused by Mycobacterium tuberculosis complex (MTBC) and can also involve nontuberculous mycobacteria (NTM). These pathogens pose significant global health challenges, particularly in developing countries. Differentiating between MTBC and NTM in clinical specimens is often difficult using conventional acid-fast staining methods, leading to an underestimation of NTM prevalence in TB-endemic regions. This study aims to identify mycobacterial species in sputum samples from patients suspected of having TB, utilizing polymerase chain reaction (PCR) assays and gene sequencing techniques. Methods: We collected 111 sputum samples from patients at Dr. Wahidin Sudirohusodo Central General Hospital, Hasanuddin University Hospital, and Makassar Community Lung Health Center. The samples were analyzed at the Clinical Microbiology Laboratory of Hasanuddin University using standard microscopy and molecular detection techniques. Descriptive statistics were employed to summarize patient demographics, infection characteristics, and outcomes. Results: We collected sputum from suspected TB patients with an average age of 50.86 years. We found 16.2% (n = 18) acid-fast bacteria in 111 patients with suspected pulmonary TB, and molecularly, we identified 17.1% (n = 19) Mycobacterium species by multiplex PCR. Three sputum samples tested positive for NTM. Phylogenetic analysis, based on 16S rRNA gene sequencing, revealed similarities between the samples and known mycobacterial species. Conclusions: The study underscores the challenges in differentiating between MTBC and NTM, highlighting the necessity for molecular diagnostic approaches. Notably, we found NTM in sputum samples from patients previously treated for TB. These findings can serve as a reference for improving diagnostic accuracy and preventing misdiagnosis of mycobacterial infections. [ABSTRACT FROM AUTHOR]

Objective: Rapid identifying and assessing the resistance patterns of multidrug-resistant tuberculosis (MDR-TB) agents, which has become a significant global public health issue, is critical for establishing and controlling the correct treatment protocols for TB. This study aims to assess the phenotypic resistance profiles of Mycobacterium tuberculosis complex (MTBC) strains isolated from clinical materials of patients with a preliminary diagnosis of tuberculosis to primary anti-tuberculosis drugs by the automated MGIT 960 SIRE system (BD Phoenix, USA) and to detect prevalent mutations in genes like rpoB, KatG, rrs, InhA, and embB linked to the development of this resistance, using the GenoType MTBDR plus test. Methods: MTBC isolates obtained from clinical samples at the mycobacteriology laboratory of Dicle University Hospital from January 2014 to August 2019 were included. The isolates were identified as MTBC isolates by an immunochromatographic antigen (Ag) test- MPT64 (BD, Poland) and found to be resistant to primary anti-tuberculosis drugs by the automated BACTEC MGIT 960 system. Among 69 isolates, 32 (46.4%) were multidrug resistant (MDR), 34 (49.3%) were resistant to isoniazid (INH) and one or more of the primary anti-tuberculosis drugs, and three (4.3%) were resistant to rifampin (RIF). Results: TCG/TTG mutation at codon 531 of rpoB gene was the common mutation responsible for resistance in 19 (57.6%) RIF resistant isolates, AGC/ACC mutation in katG315 gene region in 14 (46.7%) INH resistant isolates and C/T mutations at position -15 of inhA gene region in 8 (26.7%) INH resistant isolates. Conclusion: The genotypic test (GenoType MTBDR plus) used to identify mutations associated with resistance to primary anti- tuberculosis drugs revealed that the most common mutations linked to rifampicin resistance were the TCG/TTG mutation located at codon 531 of the rpoB gene, the AGC/ACC (Ser315Thr) mutation located at codon 315 of the katG gene, and C/T mutations at position -15 in the inhA gene region. The AGC/ACC (Ser315Thr) mutation at codon 315 of the katG gene and C/T mutations at position -15 of the inhA gene region were identified as the most common contributors to resistance, aligning with results from earlier studies on the subject. [ABSTRACT FROM AUTHOR]

The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases. Key points: • Panel of mAbs for differentiating MTB versus NTM • IC strips for diagnosing MTBC and NTM after the BACTEC MGIT • Combined detection of MTP64 and Ag85B enhances diagnostic accuracy [ABSTRACT FROM AUTHOR]

Background: The respiratory chain plays a key role in the growth of Mycobacterium tuberculosis complex (MTBC). However, the exact regulatory mechanisms of this system still need to be elucidated, and only a few studies have investigated the impact of genetic mutations within the respiratory chain on MTBC transmission. This study aims to explore the impact of respiratory chain gene mutations on the global spread of MTBC. Results: A total of 13,402 isolates of MTBC were included in this study. The majority of the isolates (n = 6,382, 47.62%) belonged to lineage 4, followed by lineage 2 (n = 5,123, 38.23%). Our findings revealed significant associations between Single Nucleotide Polymorphisms (SNPs) of specific genes and transmission clusters. These SNPs include Rv0087 (hycE, G178T), Rv1307 (atpH, C650T), Rv2195 (qcrA, G181C), Rv2196 (qcrB, G1250T), Rv3145 (nuoA, C35T), Rv3149 (nuoE, G121C), Rv3150 (nuoF, G700A), Rv3151 (nuoG, A1810G), Rv3152 (nuoH, G493A), and Rv3157 (nuoM, A1243G). Furthermore, our results showed that the SNPs of atpH C73G, atpA G271C, qcrA G181C, nuoJ G115A, nuoM G772A, and nuoN G1084T were positively correlated with cross-country transmission clades and cross-regional transmission clades. Conclusions: Our study uncovered an association between mutations in respiratory chain genes and the transmission of MTBC. This important finding provides new insights for future research and will help to further explore new mechanisms of MTBC pathogenicity. By uncovering this association, we gain a more complete understanding of the processes by which MTBC increases virulence and spread, providing potential targets and strategies for preventing and treating tuberculosis. [ABSTRACT FROM AUTHOR]

India is renowned for its complex megadiverse ecosystems and abundant biodiversity. Bovine tuberculosis (bTB) often remains synonymous with Mycobacterium bovis infection in cattle. The domain of tuberculosis (TB) among wild animals, induced by members of the Mycobacterium tuberculosis complex organisms (MTBC), is often underexplored and underreported in India. Within this context, instances of wild animal tuberculosis (wTB) have manifested across both captive and free-roaming animals. The sources contributing to wTB in animals can be human, animal, or environmental factors, thus illuminating the complex transmission pathways. The diagnosis of wTB continues to pose a formidable challenge, a consequence of the expansive taxonomic diversity in both the host and the pathogen. Complications inherent in acquiring samples from wildlife, the absence of standardized diagnostic protocols, limited insights into infection prevalence, and resource constraints compound diagnosis. Amidst these, adopting the comprehensive One Health paradigm surfaces as an imperative, accentuating the interconnectedness bridging human, animal, and environmental health. Recognizing key stakeholders and fostering intersectoral collaboration to provide enhanced diagnostic techniques driven by skilled personnel and advanced infrastructure play pivotal roles in a comprehensive strategy. Additionally, leveraging vaccination efforts contributes to effective control. A national wTB surveillance program is a cornerstone, ensuring an integrated and holistic approach to disease management. Through this review, we delve into the current landscape of wTB in India, unveiling its multifaceted challenges, and further explore the multifarious strategies that the One Health approach proffers in this dynamic endeavor. [ABSTRACT FROM AUTHOR]

The global rise of drug resistant tuberculosis has highlighted the need for improved diagnostic technologies that provide rapid and reliable drug resistance results. Here, we develop and validate a whole genome sequencing (WGS)-based test for identification of mycobacterium tuberculosis complex (MTB) drug resistance to rifampin, isoniazid, pyrazinamide, ethambutol, and streptomycin. Through comparative analysis of drug resistance results from WGS-based testing and phenotypic drug susceptibility testing (DST) of 38 clinical MTB isolates from patients receiving care in Los Angeles, CA, we found an overall concordance between methods of 97.4% with equivalent performance across culture media. Critically, prospective analysis of 11 isolates showed that WGS-based testing provides results an average of 36 days faster than phenotypic culture-based methods. We showcase the additional benefits of WGS data by investigating a suspected laboratory contamination event and using phylogenetic analysis to search for cryptic local transmission, finding no evidence of community spread amongst our patient population in the past six years. WGS-based testing for MTB drug resistance has the potential to greatly improve diagnosis of drug resistant MTB by accelerating turnaround time while maintaining accuracy and providing additional benefits for infection control, lab safety, and public health applications.

Abstract Background Tuberculosis (TB) remains a global public health event of great concern, however epidemic data on TB covering entire areas during the special period of the COVID-19 epidemic have rarely been reported. We compared the dissemination and multidrug-resistance patterns of Mycobacterium tuberculosis complex (MTBC) in the main urban area of Luoyang City, China (including six municipal jurisdictions) and nine county and township areas under its jurisdiction, aimed to establish the epidemiology of TB in this region and to provide reference for precision anti-TB in places with similar settings. Methods From 2020 to 2022, sputum samples were collected from 18,504 patients with confirmed, suspected and unexcluded TB in 10 designated TB medical institutions. Insertion sequence 6110 was amplified by PCR (rpoB gene detection if necessary) to confirm the presence of MTBC. PCR-positive specimens were analyzed by multicolor melting curve analysis to detect multidrug resistance. Results Among the 18,504 specimens, 2675 (14.5%) were MTBC positive. The positive rate was higher in the main urban area than in the county and township areas (29.8% vs. 10.9%, p 60 years were the largest group infected with MTBC in the main urban area, compared with individuals aged

Tuberculosis is a chronic granulomatous transmitting type of disease caused by the Mycobacterium tuberculosis complex. It can affect any part of the body, including the oral cavity. Oral tuberculosis can be primary or secondary, In oral cavity, tongue, buccal mucosa, lip, and palate may involve. Here, the authors present a case of a 50-year-old male patient of tuberculosis of palate, manifesting as a non healing ulcer. The ulcer was present in the middle part of the palate, having undermined edge and a non indurated margin. Though it was tender on palpation, there was no evidence of palatal perforation or bony erosion on radiographic examination. A chest radiograph revealed consolidation in the apex and right upper zone, and Cartridge Based Nucleic Acid Amplification Test (CBNAAT) of sputum was positive, but biopsy of the lesion could not be performed because of problem in patient’s consent. The authors took a chance and started antitubercular drugs. They observed the changes of ulcer at regular intervals. No topical medication was given for the ulcer. After taking antitubercular drugs, the condition improved rapidly and The ulcer healed completely after completing the Intensive Phase (IP) only. Tuberculosis is a transmitting and fatal disease. Early diagnosis with proper treatment can prevent complications and the transmission of the disease to others.

Boyong Wang,1,&ast; Qiaojie Wang,1,&ast; Mingzhang Li,1 Jinlong Yu,1 Feng Jiang,1 Yujie Hu,1 Geyong Guo,1 Xiaohua Chen,2 Jin Tang,3 Pei Han,1 Hao Shen1 1Department of Orthopedics, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200233, People’s Republic of China; 2Department of Infectious Diseases, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200233, People’s Republic of China; 3Clinical Laboratory, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200233, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Hao Shen, Department of Orthopedics, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200233, People’s Republic of China, Email shenhao7212@sina.comObjective: The diagnosis of tubercular orthopedic implant-associated infection (TB-IAI) is challenging. This study evaluated the value of metagenomic next-generation sequencing (mNGS) for the diagnosis of TB-IAI and developed a standardized diagnostic procedure for TB-IAI.Methods: The records of all patients with TB-IAI diagnosed and treated at our institution between December 2018 and September 2022 were retrospectively reviewed. Patient demographic characteristics, medical history, laboratory test, microbial culture, histopathology, and mNGS results, and time to diagnosis were recorded. The diagnostic efficiency of mNGS for TB-IAI was assessed by comparing the results and diagnostic time with that of other diagnostic modalities.Results: Ten patients were included in the analysis, including eight with prosthetic joint infections and two with fracture-related infections. The mNGS positivity rate was 100% (10/10), which was higher than that of TB-antibody (11%, 1/9), real-time quantitative polymerase chain reaction (22%, 2/9), T-SPOT.TB (25%, 2/8), purified protein derivative (50%, 4/8), microbial culture (50%, 5/10), and histopathology (20%, 2/10). mNGS shortened the time to diagnosis of TB-IAI. A standardized diagnostic procedure for TB-IAI was developed based on the findings.Conclusion: mNGS is useful for the diagnosis of TB-IAI. mNGS is recommended in cases where it is difficult to identify a pathogen using routine diagnostic tests. The standardized diagnostic procedure might improve TB-IAI diagnosis.Importance: TB-IAI is a rare infection, which occurs after orthopedic surgery and hard to diagnose microbiologically. mNGS is a new detection technique not yet discussed in current literature as a means for TB-IAI diagnostics. Here we describe a cohort of patients with TB-IAI diagnosed by mNGS show high efficiency of mNGS for detection of this pathology and present a clinical algorithm supplementing conventional methods for TB-IAI assessment.Keywords: metagenomic next-generation sequencing, implant-associated infection, Mycobacterium tuberculosis complex, orthopedic infection, diagnosis

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination andMALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networkswere visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, speciesspecific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB) continues to have significant economic and veterinary health impacts on cattle herds where the disease remains endemic. The continual tailoring of policies to address such maintenance requires an in-depth analysis of national data, underpinning new control strategies. In Ireland, when outbreaks occur, ancillary testing of herd mates deemed to be at the highest risk of exposure to reactors is undertaken using the interferon gamma (GIF) test. This highest risk cohort was hypothesised to be of a higher future risk despite this ancillary testing. We used a dataset from Ireland to model bovine test failure to the comparative tuberculin skin test using a survival analysis (observations: 39,248). Our primary exposure of interest was whether an animal that tested negative had a GIF test after the disclosure of infection within a herd during a bTB breakdown. There was evidence that animals with a negative GIF test during a breakdown had an increased risk of failing a test relative to other animals from the same herds without this exposure. The time to failure was 48.8% (95%CI: 38.3–57.5%) shorter for the exposed group relative to the unexposed group during a two-year follow-up period (2019–2022; time ratio: 0.51; 95%CI: 0.43–0.62; p < 0.001). The results from this study suggest that animals who were GIF-tested, having been deemed to have a higher risk of exposure, subsequently had shorter time-to-test failure periods. The absolute numbers of failure are small (only 2.5% of animals go on to fail during 2-year follow-up). Importantly, however, a high proportion of these high-risk herds included in the dataset failed at least one test at the follow-up (21/54 herds), impacting breakdown duration or recurrence. Such risk-informed targeting of animals could be utilised in future control policies, though further research is warranted. [ABSTRACT FROM AUTHOR]

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Background The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. Methods We designed a portable thermocycler-based real-time fluorescence loop-mediated isothermal amplification assay (cyp141-RealAmp) using six oligonucleotide primers derived from cyp141 to detect MTB. A combined number of 213 sputum samples (169 obtained from clinically diagnosed cases of pulmonary TB and 44 from a control group without tuberculosis) underwent Acid-fast bacillus (AFB) smear, culture, Xpert MTB/RIF assays, and cyp141-RealAmp assay. Results: By targeting MTB cyp141, this technique could detect as low as 10 copies/reaction within 30 min, and it was successfully rejected by other mycobacteria and other bacterial species tested. Of the 169 patients, there was no statistical difference between the detection rate of cyp141-RealAmp (92.90%, 95% CI: 89.03-96.07) and that of Xpert MTB/RIF (94.67%, 95% CI: 91.28-98.06) (P > 0.05), but both were statistically higher than that of culture (65.68%, 95% CI: 58.52-72.84) (P< 0.05) and AFB (57.40%, 95% CI: 49.94-64.86) (P< 0.05). Both cyp141-RealAmp and Xpert MTB/RIF had a specificity of 100%. Furthermore, a high concordance between cyp141-RealAmp and Xpert MTB/RIF was found (Kappa = 0.89). Conclusion: The cyp141-RealAmp assay was shown to be effective, responsive, and accurate in this study. This method offers a prospective strategy for the speedy and precise detection of MTB. [ABSTRACT FROM AUTHOR]

Tuberculosis is a chronic granulomatous transmitting type of disease caused by the Mycobacterium tuberculosis complex. It can affect any part of the body, including the oral cavity. Oral tuberculosis can be primary or secondary, In oral cavity, tongue, buccal mucosa, lip, and palate may involve. Here, the authors present a case of a 50-year-old male patient of tuberculosis of palate, manifesting as a non healing ulcer. The ulcer was present in the middle part of the palate, having undermined edge and a non indurated margin. Though it was tender on palpation, there was no evidence of palatal perforation or bony erosion on radiographic examination. A chest radiograph revealed consolidation in the apex and right upper zone, and Cartridge Based Nucleic Acid Amplification Test (CBNAAT) of sputum was positive, but biopsy of the lesion could not be performed because of problem in patient's consent. The authors took a chance and started antitubercular drugs. They observed the changes of ulcer at regular intervals. No topical medication was given for the ulcer. After taking antitubercular drugs, the condition improved rapidly and The ulcer healed completely after completing the Intensive Phase (IP) only. Tuberculosis is a transmitting and fatal disease. Early diagnosis with proper treatment can prevent complications and the transmission of the disease to others. [ABSTRACT FROM AUTHOR]

We aimed to develop a novel diagnostic method called multiplex fluorescence of loop primer upon self‐dequenching loop‐mediated isothermal amplification (mFLOS‐LAMP) for the rapid detection of Mycobacterium tuberculosis complex (MTBC). A set of specific primers was designed to target the detection of IS1081 and IS6110 genes, which are insertion sequences within the MTBC. The 110 sputum specimens collected were assessed using the established mFLOS‐LAMP method, multiplex polymerase chain reaction, Xpert MTB/RIF, and smear microscopy. The optimal reaction temperature and duration for mFLOS‐LAMP were determined to be 65°C and 30 min, respectively, by optimizing the entire system. The detection sensitivity of mFLOS‐LAMP was 6.0 × 101 CFU/mL, by Bacillus Calmette‐Guerin, and the mFLOS‐LAMP sensitivity of M. tuberculosis H37Rv genomic DNA was 500 fg, and the specificity was 100%. The sensitivity of mFLOS‐LAMP was 94.2% and the specificity was 96.6%, when Xpert MTB/RIF was used as the reference method. There was no statistically significant difference in their detection rate (χ2 = 0, P = 1.000), and the consistency was good (kappa = 0.909, P < 0.001). The receiver operating characteristic analysis yielded the maximum area under the curve of 0.954. The mFLOS‐LAMP method demonstrated high sensitivity and specificity, allowing for swift and accurate detection of MTBC. [ABSTRACT FROM AUTHOR]

Key Clinical Message: In the setting of Fournier's gangrene, atypical clinical manifestations and complications in an immunocompetent patient warrant consideration of perineal tuberculosis as a potential underlying cause. Tuberculosis cutis orificialis is a rare form of extrapulmonary tuberculosis that affects the perianal region. Fournier's gangrene is an aggressive necrotizing fasciitis that primarily involves the perianal area and external genitalia. A previously healthy 38‐year‐old man presented with a left perianal abscess. His condition deteriorated, leading to septic shock and multiorgan dysfunction syndrome. A CT scan displayed extensive necrotizing fasciitis. Treatment included broad‐spectrum antibiotics, numerous surgical perineal debridements, a transverse loop colostomy, and hyperbaric oxygen therapy. We believe the patient had pre‐existing asymptomatic, non‐diagnosed perianal tuberculosis, and a subsequent bacterial superinfection resulted in a perineal local abscess that progressed to severe Fournier's gangrene. The diagnosis of tuberculosis was confirmed through positive cultures and molecular identification in perineal biopsies. The patient experienced a complex clinical course with complications such as myocardial necrosis, acute respiratory distress syndrome, rhabdomyolysis with severe critical illness polyneuromyopathy and internal jugular thrombosis. Fournier's gangrene resulted in air dissection throughout the perineal fasciae, extending to the abdominal wall muscles resulting in an infected extraperitoneal spontaneous hematoma, probably caused by therapeutic anticoagulation. An extraperitoneal surgical drainage was performed. This case emphasizes the complexities in diagnosing and managing both perianal tuberculosis and Fournier's gangrene. [ABSTRACT FROM AUTHOR]

Animal tuberculosis (TB) is often maintained by multi-host communities, including livestock and wildlife. Quantitative studies of such communities require estimating the true prevalence of TB, correcting the apparent prevalence by the diagnostic sensitivity (Se) and specificity (Sp) of the test. The goal of this study was to lay the foundations for estimating the true prevalence of TB in wild ungulate populations (wild boar and two cervids: red deer and fallow deer). We used Bayesian latent class models to assess the Se and Sp of gross pathology, IS6110 real-time PCR in tissues, bacteriological culture, and P22 indirect ELISA. We analyzed 308 harvested wild ungulates (211 wild boar and 97 cervids: 92 red deer and 5 fallow deer). The Se of bacteriological culture (80.4%, CI95 61.0–96.3%) and gross pathology (87.9%, CI95 69.5–99.9%) was reasonably good in wild boar. These tests showed lower Se in cervids: 60.2% (CI95 38.3–82.3%) for bacteriological culture and 81.5% (CI95 63.6–96.2%) for gross pathology. The Se of the real-time PCR was low (50.7% in wild boar and 53.0% in cervids). These tests showed Sp between 95.2 and 99.1% in both taxa. The P22 ELISA performed reasonably well in wild boar (Se = 71.9%, CI95 59.2–83.4%; Sp = 98.8%, CI95 96.9–99.9%) but lacked Sp in cervids (Se = 77.1%, CI95 62.9–89.7%; Sp = 74.5%, CI95 65.7–83.3%). The real-time PCR in wild boar and cervids and bacteriological culture in cervids tended to show higher Se in low-prevalence populations, possibly due to a higher proportion of early-stage TB lesions. In cervids, the parallel interpretation of gross pathology and bacteriological culture significantly improved the diagnostic performance (Se = 93.1%, CI95 84.7–98.9%; Sp = 92.9%, CI95 86.0–98.3%). Our results allow the estimation of true prevalence from the results of a single diagnostic test applied to harvested wild boar, red deer, and fallow deer, paving the way for more precise quantitative ecological studies of the multi-host TB maintenance community.

Background/Aim: Tuberculosis remains a major global health problem with a high morbidity and mortality rate, approximately a quarter of the population is infected with tuberculosis. Drug susceptibility testing is an essential tool for identifying and managing drug-resistant tuberculosis. This study was conducted to evaluate the drug susceptibility pattern of Mycobacterium tuberculosis complex strains isolated from a university hospital. Methods: A total of 10900 samples sent to the microbiology laboratory with the suspicion of tuberculosis clinically between January 2018 and January 2022 were analyzed retrospectively. The automated BACTEC MGIT 960 (Becton Dickinson, USA) was used for sample culture and susceptibility testing. The obtained data were statistically analyzed with the Statistical Package for Social Sciences (SPSS version 20). Results: Out of the 154 isolated positive samples, males and females constituted equal parts of the study population (50%). The majority of tuberculosis cases were in the age group 56–75 years (42.2%), Pulmonary TB was detected in (90.3%) of the patients, while extrapulmonary TB cases were observed in (9.7%). As a result of susceptibility studies on positive samples, isoniazid resistance was 5.2%; streptomycin resistance 1.3%; ethambutol resistance was detected at a rate of 0.6%, while no rifampicin resistant sample was found. Both streptomycin and isoniazid resistance were seen together in 1.3% of the samples. Conclusion: A similar resistance pattern of the first-line antituberculosis drugs was observed in other studies conducted in different provinces of Turkey. The absence of multi-drug resistant and extensively drug-resistant tuberculosis in our study indicates that the tuberculosis surveillance program implemented in our region was successful.

Abstract The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain (“matched infection”) or with strains representative of other MTBC lineages (“mismatched infection”). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1β, and IL-1β compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host–pathogen interaction in human tuberculosis.

Abstract Background Drug-resistant tuberculosis (TB) is a major threat to global public health. Whole-genome sequencing (WGS) is a useful tool for species identification and drug resistance prediction, and many clinical laboratories are transitioning to WGS as a routine diagnostic tool. However, user-friendly and high-confidence automated bioinformatics tools are needed to rapidly identify M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), detect drug resistance, and further guide treatment options. Results We developed GenoMycAnalyzer, a web-based software that integrates functions for identifying MTBC and NTM species, lineage and spoligotype prediction, variant calling, annotation, drug-resistance determination, and data visualization. The accuracy of GenoMycAnalyzer for genotypic drug susceptibility testing (gDST) was evaluated using 5,473 MTBC isolates that underwent phenotypic DST (pDST). The GenoMycAnalyzer database was built to predict the gDST for 15 antituberculosis drugs using the World Health Organization mutational catalogue. Compared to pDST, the sensitivity of drug susceptibilities by the GenoMycAnalyzer for first-line drugs ranged from 95.9% for rifampicin (95% CI 94.8–96.7%) to 79.6% for pyrazinamide (95% CI 76.9–82.2%), whereas those for second-line drugs ranged from 98.2% for levofloxacin (95% CI 90.1–100.0%) to 74.9% for capreomycin (95% CI 69.3–80.0%). Notably, the integration of large deletions of the four resistance-conferring genes increased gDST sensitivity. The specificity of drug susceptibilities by the GenoMycAnalyzer ranged from 98.7% for amikacin (95% CI 97.8–99.3%) to 79.5% for ethionamide (95% CI 76.4–82.3%). The incorporated Kraken2 software identified 1,284 mycobacterial species with an accuracy of 98.8%. GenoMycAnalyzer also perfectly predicted lineages for 1,935 MTBC and spoligotypes for 54 MTBC. Conclusions GenoMycAnalyzer offers both web-based and graphical user interfaces, which can help biologists with limited access to high-performance computing systems or limited bioinformatics skills. By streamlining the interpretation of WGS data, the GenoMycAnalyzer has the potential to significantly impact TB management and contribute to global efforts to combat this infectious disease. GenoMycAnalyzer is available at http://www.mycochase.org .

ABSTRACT: Objectives: The characteristic and performance of Broth microdilution (BMD) plates for drug susceptibility of Mycobacterium tuberculosis have not been systematically evaluated in China. This study was designed to review the key information and assess the performance of BMD plates by analysis of proficiency testing results. Methods: We retrospectively analysed the proficiency testing results of phenotypic drug susceptibility testing (PT-DST) of 45 laboratories using BMD plates in China in 2021. Critical information, such as drug layout, concentration range of each drug, plate storage conditions and duration, operating procedures, and interpretation criteria for binary results were compared. The performance was also analysed. Results: Eight types of BMD plates produced by four manufactures were reported. The drug layout, number of drugs on plates, and concentration range varied a lot between different plates. The total sensitivity and specificity of BMD plates for drug susceptibility of Mycobacterium tuberculosis to ten drugs (isoniazid (INH), rifampin (RIF), kanamycin (KAM), amikacin (AM), levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), linezolid (LZD), clofazimine (CFZ), and delamanid (DLM)) were 93.9% (95% CI 92.–94.9) and 99.1% (95% CI 98.8–99.3), respectively. The lowest sensitivity was 84.8% (95% CI 80.3–88.4) for LFX and 86.4% (95% CI 82.5–89.6) for MFX, or 87.5% (95% CI 84.2–90.2) for Y1 plate and 87.9% (95% CI 83.5–91.1) for T plate. The lowest specificity was 94.4% (95% CI 91.4–96.4) for DLM, or 97.9% (95% CI 96.8–98.7) for B3 plate. Conclusion: Commercial BMD plates in China showed varied drug layouts and operational procedures, indicating the urgency of standardization. The lower performance for some drugs showed the low quality of the plates utilized or lack of proficiency of lab staffs in operating and interpreting results.

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 IS 6110, which is widely used as a diagnostic marker. IS 6110 transposition drives genetic modifications in MTBC, imparting genome plasticity and profound biological consequences. While IS 6110 insertions are customarily found in the MTBC genomes, the evolutionary trajectory of strains seems to correlate with the number of IS 6110 copies, indicating enhanced adaptability with increasing copy numbers. Here, we present a comprehensive analysis of IS 6110 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 IS 6110 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 IS 6110 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 IS 6110 insertions, their preferential insertion regions, and their impact on metabolic processes and pathways, providing valuable knowledge on the genetic changes underpinning IS 6110 transposition in M. orygis. [ABSTRACT FROM AUTHOR]

Background/Aim: Tuberculosis (TB) remains a major global health problem with a high morbidity and mortality rate, approximately a quarter of the population is infected with tuberculosis. Drug susceptibility testing is an essential tool to identify and manage drug-resistant tuberculosis. This study was conducted to evaluate the drug susceptibility pattern of Mycobacterium tuberculosis complex strains isolated from a university hospital. Methods: A total of 10900 samples sent to the microbiology laboratory with the suspicion of tuberculosis clinically between January 2018 and January 2022 were analyzed retrospectively. The automated BACTEC MGIT 960 (Becton Dickinson, USA) was used for sample culture and susceptibility testing. The obtained data were statistically analyzed with the Statistical Package for Social Sciences (SPSS version 20). Results: Out of the 154 isolated positive samples, males and females constituted equal parts of the study population (50%). The majority of tuberculosis cases were in the age group of 56-75 years (42.2%). Pulmonary tuberculosis was detected in 139 (90.3%) of the patients while extrapulmonary TB cases were observed in 15 (9.7%) patients. As a result of susceptibility studies on positive samples, isoniazid resistance was 5.2%; streptomycin resistance 1.3%; ethambutol resistance was detected at a rate of 0.6% while no rifampicin resistant sample was found. Both streptomycin and isoniazid resistance were seen together in 1.3% of the samples. Conclusion: A similar resistance pattern of the first-line antituberculosis drugs was observed in other studies conducted in different provinces of Turkiye. The absence of multi-drug resistant and extensively drug-resistant tuberculosis in our study indicates that the tuberculosis surveillance program implemented in our region was successful. [ABSTRACT FROM AUTHOR]

Introduction: Bovine tuberculosis (bTB) caused by Mycobacterium tuberculosis 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 IS6110 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. Methods: The fresh LNs (N=100) were collected each from a different cattle carcass at the slaughterhouse. The limit of detection of ddPCR-IS6110 was set to 101 copies per 20 ml reaction. Results: DdPCR-IS6110 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-IS6110-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-IS6110 disclosed as positive 9 samples negative to reference-standard. Discussion: DdPCR-IS6110 proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method. [ABSTRACT FROM AUTHOR]

The diagnosis and treatment of patients with mendelian susceptibility to mycobacterial disease (MSMD) pose consistent challenges due to the diverse infection spectrum observed in this population. Common clinical manifestations include Bacillus Calmette-Guérin vaccine (BCG) complications in countries where routine BCG vaccination is practiced, while in non-BCG-vaccinating countries, Non-Tuberculous Mycobacteria (NTM) is prevalent. In tuberculosis-endemic regions, Mycobacterium tuberculosis (MTB) has a high prevalence, along with other intracellular organisms. Isolating these organisms presents a significant challenge, and treatment is often initiated without confirming the specific species. This review primarily focuses on the methods and challenges associated with diagnosing and treating MSMD patients. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host–pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein–protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as “Complement activation, alternative pathway” and “Tricarboxylic acid cycle”, which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as “Natural killer cell degranulation” in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

Background: 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. Methods: 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, MTBDRplus VER 2.0, and MTBDRsl 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. Results: 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. Conclusions: 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.

Key Clinical Message In the setting of Fournier's gangrene, atypical clinical manifestations and complications in an immunocompetent patient warrant consideration of perineal tuberculosis as a potential underlying cause. Abstract Tuberculosis cutis orificialis is a rare form of extrapulmonary tuberculosis that affects the perianal region. Fournier's gangrene is an aggressive necrotizing fasciitis that primarily involves the perianal area and external genitalia. A previously healthy 38‐year‐old man presented with a left perianal abscess. His condition deteriorated, leading to septic shock and multiorgan dysfunction syndrome. A CT scan displayed extensive necrotizing fasciitis. Treatment included broad‐spectrum antibiotics, numerous surgical perineal debridements, a transverse loop colostomy, and hyperbaric oxygen therapy. We believe the patient had pre‐existing asymptomatic, non‐diagnosed perianal tuberculosis, and a subsequent bacterial superinfection resulted in a perineal local abscess that progressed to severe Fournier's gangrene. The diagnosis of tuberculosis was confirmed through positive cultures and molecular identification in perineal biopsies. The patient experienced a complex clinical course with complications such as myocardial necrosis, acute respiratory distress syndrome, rhabdomyolysis with severe critical illness polyneuromyopathy and internal jugular thrombosis. Fournier's gangrene resulted in air dissection throughout the perineal fasciae, extending to the abdominal wall muscles resulting in an infected extraperitoneal spontaneous hematoma, probably caused by therapeutic anticoagulation. An extraperitoneal surgical drainage was performed. This case emphasizes the complexities in diagnosing and managing both perianal tuberculosis and Fournier's gangrene.

IntroductionBovine tuberculosis (bTB) caused by Mycobacterium tuberculosis 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 IS6110 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. MethodsThe fresh LNs (N=100) were collected each from a different cattle carcass at the slaughterhouse. The limit of detection of ddPCR-IS6110 was set to 101 copies per 20 μl reaction.ResultsDdPCR-IS6110 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-IS6110-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-IS6110 disclosed as positive 9 samples negative to reference-standard. DiscussionDdPCR-IS6110 proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method.

Introduction: Understanding the epidemiological and clinical characteristics of different tuberculosis strains is crucial for developing improved diagnostic tools, drugs, and vaccines for tuberculosis management. This study aimed to investigate the molecular epidemiology of Mycobacterium tuberculosis using spoligotyping, a widely used molecular typing method, to understand the genetic diversity and transmission dynamics of M. tuberculosis, on isolates obtained from patients with pulmonary tuberculosis in central Kerala. Methods: In a prospective study at a tertiary care hospital, 404 respiratory specimens from patients with symptoms suggestive of TB were collected. Specimens underwent Ziehl-Neelsen staining, culture in liquid (BD BACTEC™ MGIT™) and solid (Lowenstein-Jensen) media, and standard drug susceptibility testing with the MGIT system. Molecular analysis involved conventional PCR amplification of genomic DNA to generate sufficient genetic material for analysis, using species-specific and primers targeting the direct repeat region, followed by spoligotyping to assess the genetic diversity of the M. tuberculosis strains. Results: Out of 404 samples from individuals with suspected pulmonary TB, Mycobacteria were cultured from 48 [11.9%] of the samples. Amongst the 48 culture-positive M. tuberculosis isolates, 20 (41.66%) were sensitive to all five first-line anti-TB drugs, and 3 (6.2%) were resistant to all five drugs. Spoligotyping of the 47 isolates showed that 36.1% [n=17] of the isolates belonged to the M. tuberculosis EAI3 (East African-Indian) family, followed by 27.6% (n=13) M. tuberculosis EAI5 and 21.2% (n=10) M. tuberculosis CAS (Central Asia). Other families observed in this study, although less prevalent, were M. tuberculosis Beijing, 8.5% (n=4), family 33, 4.3% (n=2), and Mycobacterium bovis-BCG family, 2.1% (n=1). Conclusion: This study explored the genetic diversity and distribution of circulating M. tuberculosis strains in central Kerala. Genotyping M. tuberculosis strains provides valuable insights into TB transmission and progression, which can inform the development of effective public health control strategies. [ABSTRACT FROM AUTHOR]

Wild small rodents are considered the natural reservoirs of Mycobacterium microti, a member of the Mycobacterium tuberculosis complex (MTBC) that can cause tuberculosis (TB) in humans and animals, as well as interfere with current tuberculosis eradication plans in livestock. A cross-sectional study was carried out in the Catalan Pyrenees (Iberian Peninsula) in an area where M. microti was previously isolated from wild boars, to evaluate the role of micromammals in the epidemiology of this outbreak. A total of 350 wild rodents were necropsied (306 Murinae and 44 Arvicolinae) in spring and autumn during two consecutive natural years. Tissues were analyzed by histopathology to look for TB-like lesions and by qPCR and culture to detect MTBC. Sera were analyzed by MTBC-specific ELISA. No evidence of TB infection in wild rodents was confirmed. Results suggest that small rodents did not play a role in the epidemiology of M. microti in the area. The source of this mycobacterium remains unknown, but previous detections of M. microti in various species in southern France suggest the movements of wild boars across the French Pyrenees as the most likely origin of the outbreak detected in the Iberian Peninsula. [ABSTRACT FROM AUTHOR]

Abstract The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1–26.3% (Bovigam: ODbovis–ODPBS ≥ 0.1 and ODbovis–ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98–99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status.

Bovine tuberculosis (bTB) continues to have significant economic and veterinary health impacts on cattle herds where the disease remains endemic. The continual tailoring of policies to address such maintenance requires an in-depth analysis of national data, underpinning new control strategies. In Ireland, when outbreaks occur, ancillary testing of herd mates deemed to be at the highest risk of exposure to reactors is undertaken using the interferon gamma (GIF) test. This highest risk cohort was hypothesised to be of a higher future risk despite this ancillary testing. We used a dataset from Ireland to model bovine test failure to the comparative tuberculin skin test using a survival analysis (observations: 39,248). Our primary exposure of interest was whether an animal that tested negative had a GIF test after the disclosure of infection within a herd during a bTB breakdown. There was evidence that animals with a negative GIF test during a breakdown had an increased risk of failing a test relative to other animals from the same herds without this exposure. The time to failure was 48.8% (95%CI: 38.3–57.5%) shorter for the exposed group relative to the unexposed group during a two-year follow-up period (2019–2022; time ratio: 0.51; 95%CI: 0.43–0.62; p < 0.001). The results from this study suggest that animals who were GIF-tested, having been deemed to have a higher risk of exposure, subsequently had shorter time-to-test failure periods. The absolute numbers of failure are small (only 2.5% of animals go on to fail during 2-year follow-up). Importantly, however, a high proportion of these high-risk herds included in the dataset failed at least one test at the follow-up (21/54 herds), impacting breakdown duration or recurrence. Such risk-informed targeting of animals could be utilised in future control policies, though further research is warranted.

Tuberculosis (TB) is a leading infectious killer worldwide. Over two-thirds of new TB diagnoses in the United States occur among first-generation immigrants, especially within a year of migration. Hodgkin lymphoma (HL) accounts for a minority of lymphoma cases but presents similarly to disseminated or extrapulmonary TB. Clinical overlap between TB and HL increases patient risk of misdiagnosis. Concomitant presentation of both diseases is not uncommon but infrequently reported. We present a case of isoniazid-resistant TB with progressively worsening lymphadenopathy and splenomegaly despite appropriate TB treatment. The patient was diagnosed with HL following PET/CT and axillary lymph node biopsy.

Introduction: The population structure of Mycobacterium tuberculosis complex (MTBC) in Ethiopia is diverse but dominated by Euro-American (Lineage 4) and East-African-Indian (Lineage 3) lineages. The objective of this study was to describe the genetic diversity of MTBC isolates in Central, Eastern and Southeastern Ethiopia. Methods: A total of 223 MTBC culture isolates obtained from patients referred to Adama and Harar TB reference laboratories were spoligotyped. Demographic and clinical characteristics were collected. Results: Six major lineages: Euro-American (Lineage 4), East-African-Indian (Lineage 3), East Asian (Lineage 2), Indo-Oceanic (Lineage 1), Mycobacterium africanum (Lineage 5 and Lineage 6) and Ethiopian (Lineage 7) were identified. The majority (94.6 %) of the isolates were Euro-American and East-African-Indian, with proportions of 75.3 % and 19.3 %, respectively. Overall, 77 different spoligotype patterns were identified of which 42 were registered in the SITVIT2 database. Of these, 27 spoligotypes were unique, while 15 were clustered with 2–49 isolates. SIT149/T3_ETH (n = 49), SIT53/T1 (n = 33), SIT21/CAS1_Kili (n = 24) and SIT41/Turkey (n = 11) were the dominant spoligotypes. A rare Beijing spoligotype pattern, SIT541, has also been identified in Eastern Ethiopia. The overall clustering rate of sub-lineages with known SIT was 71.3 %. Age group (25–34) was significantly associated with clustering. Conclusion: We found a heterogeneous population structure of MTBC dominated by T and CAS families, and the Euro-American lineage. The identification of the Beijing strain, particularly the rare SIT541 spoligotype in Eastern Ethiopia, warrants a heightened surveillance plan, as little is known about this genotype. A large-scale investigation utilizing a tool with superior discriminatory power, such as whole genome sequencing, is necessary to gain a thorough understanding of the genetic diversity of MTBC in the nation, which would help direct the overall control efforts.

Background: Mycobacterium avium complex (MAC) is connected to human immunosuppressive diseases, including HIV-AIDS, and may pose a zoonotic threat. MAC causes lymphadenopathy in children, respiratory infection in adults, and generalized infection in immunocompromised individuals. Infection with nontuberculous Mycobacteria (NTM) in humans is now primarily brought on by MAC. Recently, MAC members have emerged as pathogenic organisms for animals and humans. While dogs are generally resistant to mycobacterial infections, there have been some cases of infection that result in systemic or disseminated diseases. The organisms can be transmitted to dogs through oral contact, and their faeces can be a possible source of infection for dog owners. It is important to note that this ailment is zoonotic, especially if infected pet dogs are in prolonged contact with their humans. Aims: The study was planned to demonstrate the occurrence of MAC organisms and other Mycobacteria in dogs associated with lymphadenopathy cases with special emphasis on lymphadenitis. Methods: A total of 123 samples (100 lymph node aspirates, 15 lymph node tissues, and 8 blood samples) from 83 dogs suspected of lymphadenitis accompanied by gastroenteritis, chronic skin infections, immunosuppression, chronic pulmonary diseases, and other chronic undiagnosed diseases were studied. The samples were processed for cytological and microscopic examination by Ziehl-Neelsen staining. Following the decontamination procedure, the aspiration and lymph node tissue samples were inoculated into Middlebrook 7H11 media for up to 8 weeks. The aspirated material was also directly used for molecular detection by triplex-nested polymerase chain reaction (nPCR) assay. Results: A cytological study revealed pyogranulomatous inflammation of the lymph node tissue. Impression smears from lymph node tissues displayed the presence of acid-fast organisms. Out of 83 cases of dogs, 8 were found to be positive for Mycobacterium spp. Among those 8 positive cases, 3 were confirmed to belong to MAC, and 5 belonged to the Mycobacterium tuberculosis complex (MTB complex). Conclusion: MAC and MTB are the underestimated bacteria that could be the causative agents of lymphadenitis in animals. [ABSTRACT FROM AUTHOR]