Mycobacterium Tuberculosis Complex DNA Research Articles

Metagenomic and paleopathological analyses of a historic documented collection explore ancient dental calculus as a diagnostic tool

Dental calculus is a microbial biofilm that contains biomolecules from oral commensals and pathogens, including those potentially related to cause of death (CoD). To assess the utility of calculus as a diagnostically informative substrate, in conjunction with paleopathological analysis, calculus samples from 39 individuals in the Smithsonian Institution’s Robert J. Terry Collection with CoDs of either syphilis or tuberculosis were assessed via shotgun metagenomic sequencing for the presence of Treponema pallidum subsp. pallidum and Mycobacterium tuberculosis complex (MTBC) DNA. Paleopathological analysis revealed that frequencies of skeletal lesions associated with these diseases were partially inconsistent with diagnostic criteria. Although recovery of T. p. pallidum DNA from individuals with a syphilis CoD was elusive, MTBC DNA was identified in at least one individual with a tuberculosis CoD. The authenticity of MTBC DNA was confirmed using targeted quantitative PCR assays, MTBC genome enrichment, and in silico bioinformatic analyses; however, the lineage of the MTBC strain present could not be determined. Overall, our study highlights the utility of dental calculus for molecular detection of tuberculosis in the archaeological record and underscores the effect of museum preparation techniques and extensive handling on pathogen DNA preservation in skeletal collections.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone

ObjectivesWe evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. MethodsFTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. ResultsOf the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DiscussionFTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

Targeted sequencing from cerebrospinal fluid for rapid identification of drug-resistant tuberculous meningitis.

Mortality from tuberculous meningitis (TBM) remains around 30%, with most deaths occurring within 2 months of starting treatment. Mortality from drug-resistant strains is higher still, making early detection of drug resistance (DR) essential. Targeted next-generation sequencing (tNGS) produces high read depths, allowing the detection of DR-associated alleles with low frequencies. We applied Deeplex Myc-TB-a tNGS assay-to cerebrospinal fluid (CSF) samples from 72 adults with microbiologically confirmed TBM and compared its genomic drug susceptibility predictions to a composite reference standard of phenotypic susceptibility testing (pDST) and whole genome sequencing, as well as to clinical outcomes. Deeplex detected Mycobacterium tuberculosis complex DNA in 24/72 (33.3%) CSF samples and generated full DR reports for 22/24 (91.7%). The read depth generated by Deeplex correlated with semi-quantitative results from MTB/RIF Xpert. Alleles with <20% frequency were seen at canonical loci associated with first-line DR. Disregarding these low-frequency alleles, Deeplex had 100% concordance with the composite reference standard for all drugs except pyrazinamide and streptomycin. Three patients had positive CSF cultures after 30 days of treatment; reference tests and Deeplex identified isoniazid resistance in two, and Deeplex alone identified low-frequency rifampin resistance alleles in one. Five patients died, of whom one had pDST-identified pyrazinamide resistance. tNGS on CSF can rapidly and accurately detect drug-resistant TBM, but its application is limited to those with higher bacterial loads. In those with lower bacterial burdens, alternative approaches need to be developed for both diagnosis and resistance detection.

Droplet digital PCR as alternative to microbiological culture for Mycobacterium tuberculosis complex detection in bovine lymph node tissue samples.

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. 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 μl reaction. 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. DdPCR-IS6110 proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method.

Bioarchaeological investigation of individuals with suspected multibacillary leprosy from the mediaeval leprosarium of St Mary Magdalen, Winchester, Hampshire, UK.

Introduction. We have examined four burials from the St Mary Magdalen mediaeval leprosarium cemetery in Winchester, Hampshire, UK. One (Sk.8) was a male child, two (Sk.45 and Sk.52) were adolescent females and the fourth (Sk.512) was an adult male. The cemetery was in use between the 10th and 12th centuries. All showed skeletal lesions of leprosy. Additionally, one of the two females (Sk.45) had lesions suggestive of multi-cystic tuberculosis and the second (Sk.52) of leprogenic odontodysplasia (LO), a rare malformation of the roots of the permanent maxillary incisors.Gap statement. Relatively little is known of the manifestations of lepromatous leprosy (LL) in younger individuals from the archaeological record.Aims and Methodology. To address this, we have used ancient DNA testing and osteological examination of the individuals, supplemented with X-ray and microcomputed tomography (micro-CT) scan as necessary to assess the disease status.Results and Conclusions. The presence of Mycobacterium leprae DNA was confirmed in both females, and genotyping showed SNP type 3I-1 strains but with a clear genotypic variation. We could not confirm Mycobacterium tuberculosis complex DNA in the female individual SK.45. High levels of M. leprae DNA were found within the pulp cavities of four maxillary teeth from the male child (Sk.8) with LO, consistent with the theory that the replication of M. leprae in alveolar bone may interfere with root formation at key stages of development. We report our biomolecular findings in these individuals and review the evidence this site has contributed to our knowledge of mediaeval leprosy.

Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection.

This study aimed to evaluate the accuracy of detecting drug-resistant Mycobacterium tuberculosis 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 IS6110 and the mtp40 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 IS6110 and mtp40 single-step PCR revealed the IS6110 in 27 of the 48 sputum samples, while the mtp40 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.

IMPROVING THE DIAGNOSIS OF SPINAL TUBERCULOSIS AND OUR UNDERSTANDING OF ITS PATHOPHYSIOLOGY AND INTERACTION WITH HIV-1 CO-INFECTION

Specific and rapid detection methods for spinal tuberculosis, with sufficient sensitivity in HIV-1 co-infected individuals, are needed, to ensure early initiation of appropriate treatment to prevent physical disability and neurological fallout. In addition, understanding the systemic and local pathophysiology of spinal tuberculosis, and its interaction with HIV-1 infection, is crucial to guide future therapeutic interventions.We prospectively enrolled adult patients presenting with signs and symptoms of suspected spinal tuberculosis, at Groote Schuur Hospital, between November 2020 and December 2021. TB diagnostic testing was performed on open and CT-guided spinal biopsies using Xpert MTB/RIF Ultra compared to gold standards TB culture and histology. A highly sensitive droplet digital PCR assay for detecting and quantifying Mycobacterium tuberculosis complex (MTBC) and HIV-1 DNA was tested. Plasma inflammatory proteins were measured to assess systemic inflammation.Xpert Ultra had a high sensitivity of 94.7% and specificity of 100% for STB against TB culture and histology in both open and CT-guided biopsy samples. The ddPCR assay confirmed TB detection in 94% of patients with positive Xpert Ultra results. Four patients with negative TB diagnostic results had MTBC DNA detected by ddPCR. HIV-1 DNA was detected in the spinal tissues from all HIV-1-infected patients. MTBC DNA levels were significantly higher in HIV-1-co-infected spinal tissue samples (p&lt; 0.01). We identified four biomarkers significantly associated with higher bacterial burden at the disease site (p&lt; 0.01).Xpert Ultra and MTBC ddPCR improve the detection of STB. DdPCR can be utilized as an additional, highly sensitive tool for detecting and quantifying Mtb, in pathological samples that may be paucibacillary. These findings provide novel diagnostic and pathophysiologic insight into STB, in the context of HIV-1 infection, and provide rationale to include these tests in hospital and research settings for patients from communities burdened by TB and HIV-1.

Tenosynovitis of the Flexor Digitorum and Flexor Carpi Caused by &lt;i&gt;Mycobacterium Tuberculosis&lt;/i&gt;: Case Report and Review

Background. Mycobacterial tenosynovitis of the hand and forearm is an extremely rare form of extrapulmonary tuberculosis, which can lead to permanent disability of patients.&#x0D; Case presentation. We present a rare clinical case of generalized tenosynovitis of the tendons of all hand and finger flexors of the right upper extremity caused by Mycobacterium tuberculosis (MBT). Patient complained of a lump on the palmar surface of the right hand with periodic spontaneous discharge in the form of rice grains. MRI revealed a peritendinal synovial cystic mass on the volar surface of the hand and wrist joint with the presence of multiple chondromal bodies, tendinitis and tenosynovitis of the flexor of IV and V fingers. Removal of the neoplasm, pathological tissues, and total synovectomy were performed. Intraoperative material was tested using PCR. DNA of Mycobacterium tuberculosis complex was detected.&#x0D; Conclusion. Presented case report demonstrates the importance of preoperative MRI examination in tenosynovitis of unknown etiology and the necessity of histological and bacteriological examination of intraoperative material. Transportation of obtained tissues for examination should be performed without the use of formalin in order to exclude false negative results. One should be vigilant against nonspecific infections, including Mycobacterium tuberculosis, when treating patients with immunocompromising diseases. The presence of limb neuropathy, instrumental signs of the second or the third stage of tuberculous tenosynovitis according to Kanavel classification are indications for surgical treatment.

Unlocking environmental contamination of animal tuberculosis hotspots with viable mycobacteria at the intersection of flow cytometry, PCR, and ecological modelling

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC), circulates in multi-host mammal communities. While interactions between different host species are mainly indirect, current knowledge postulates interspecific transmission is favored by animal contact with natural substrates contaminated with droplets and fluids from infected animals. However, methodological constraints have severely hampered monitoring of MTBC outside its hosts and the subsequent validation of this hypothesis.In this work, we aimed to evaluate the extent to which environmental contamination with M. bovis occurs in an endemic animal TB setting, taking advantage of a new real-time monitoring tool we recently developed to quantify the proportion of viable and dormant MTBC cell fractions in environmental matrices. Sixty-five natural substrates were collected nearby the International Tagus Natural Park region, in the epidemiological TB risk area in Portugal. These included sediments, sludge, water, and food deployed at unfenced feeding stations. The tripartite workflow included detection, quantification, and sorting of different M. bovis cell populations: total, viable, and dormant. Real-time PCR targeting IS6110 to detect MTBC DNA was performed in parallel. The majority of samples (54 %) contained metabolically active or dormant MTBC cells. Sludge samples had a higher burden of total MTBC cells and a high concentration of viable cells (2.3 × 104 cells/g). Ecological modelling informed by climate, land use, livestock and human disturbance data suggested eucalyptus forest and pasture cover as potential major factors affecting the occurrence of viable MTBC cells in natural matrices.Our study demonstrates, for the first time, the widespread environmental contamination of animal TB hotspots with viable MTBC bacteria and with dormant MTBC cells that are able to recover metabolic activity. Further, we show that viable MTBC cell load in natural substrates is superior to the estimated minimum infective dose, providing real-time insights into the potential magnitude of environmental contamination for indirect TB transmission.

Xpert MTB/RIF Ultra Trace Results: Decision Support for the Treatment of Extrapulmonary Tuberculosis in Low TB Burden Countries.

Extrapulmonary tuberculosis (EPTB) can be difficult to diagnose, especially in severe forms. The Xpert MTB/RIF Ultra test introduced an additional category called trace to reference very small amounts of Mycobacterium tuberculosis complex (MTBC) DNA. The objective of our multicenter study was to evaluate whether the trace result on an extrapulmonary (EP) sample is a sufficient argument to consider diagnosing tuberculosis and starting treatment, even in severe cases. A retrospective, multicenter cohort study was conducted from 2018 to 2022. Patients strongly suspected of EPTB with a trace result on an EP specimen were included. Hospital records were reviewed for clinical, treatment, and paraclinical data. A total of 52 patients were included, with a severe form in 22/52 (42.3%) cases. Culture was positive for MTBC in 33/46 (71.7%) cases. Histological analysis showed granulomas in 36/45 (80.0%) cases. An Ultra trace result with a presumptive diagnosis of TB led to the decision to treat 41/52 (78.8%) patients. All patients were started on first-line anti-TB therapy (median duration of 6.1 months), with a favorable outcome in 31/35 (88.6%) patients. The presence of a small amount of MTBC genome in EPTB is a sufficient argument to treat patients across a large region of France.

Primary tuberculosis with miliary hematogenous generalization in old age

Currently, the problems of the mechanisms of immune and inflammatory response aging, which determine the mechanisms of the development of the inflammatory process in the elderly, are being actively studied. A sectional observation of senile tuberculosis in a patient K., 78 years old, who had been treated for 4 months in the University Clinical Hospital No. 4 of the First Sechenov Moscow State Medical University in 2018 is presented. After autopsy, histological, histochemical and PCR examination, the following pathoanatomical diagnosis was made: primary (senile) tuberculosis with hematogenous and lympho-vascular generalization, with a focus of caseous pneumonia in the III segment of the right lung; caseous lymphadenitis of bifurcation, thoracic and abdominal paraaortic lymph nodes; miliary and large-focal generalization in the lungs, parietal and visceral pleura on the left, spleen and liver (acid fast stain; PCR study No. 18-5935 dated 08/13/2018 (Central Research Institute of Tuberculosis) detected DNA of mycobacterium tuberculosis complex). A feature of senile tuberculosis is the course of primary tuberculosis with hematogenic generalization and predominantly exudative-necrotic tissue reaction. Its pathogenesis is mainly due to the phenomenon of "aging immune system" (immunosenescence), as well as inflammaging — features of the inflammatory response in old age. Their main characteristics are the preactivated status of the immune system even before the recognition of the antigen, characterized by a basal level of circulating cytokines, as well as a sharp weakening of the adaptive immune response in the presence of an antigen associated with impaired functioning of the immune system with a large number of memory T cells lacking naive T-cells.

Tuberculosis with isolated atelectasis: a case report

Introduction Tuberculosis is a significant health issue in developing countries, with the World Health Organization reporting over 10 million cases worldwide in 2014. While methods exist for rapid investigation of Mycobacterium tuberculosis, radiological imaging may lead to an earlier suspicion, such as presence of cavitation. In this case report, a patient with only radiological finding being atelectasis will be presented. Case Presentation An 18 years old female patient with no specific medical background history had been evaluated at an outpatient clinic with complaints of cough and exertional dyspnea. After a fluoroquinolone treatment for 7 days given for a pericardiac density in chest radiography, patient was admitted due to limited clinical response. A chest tomography was performed while the patient was under wide spectrum antibiotics and atelectasis in right lower lobe was observed. Repeated Acid-Fast Bacilli smears and sputum samples were found negative and bronchoscopy sampling did not show any findings of obstruction. Mycobacterium tuberculosis complex DNA was later detected in the lavage samples for which antituberculosis treatment was initiated with a regimen of isoniazid 5 mg/kg, pyrazinamide 25 mg/kg, ethambutol 15 mg/kg, and rifampin 10mg/kg. Discussion The diagnosis of tuberculosis, in this case, was probably masked during the AFB smear testing due to a treatment regimen of fluoroquinolone. The general radiological definition of tuberculosis primarily consists of older studies, with the expected presentation being a combination of cavitation and infiltration. Normal lung imaging results had also been described for tuberculosis regarding chest radiography, and studies have reported the presence of atelectasis in patients with tuberculosis, as seen in tomography case series; however, the isolation of atelectasis had not been specified in the studies. Conclusion Pulmonary tuberculosis may present itself with a myriad of radiological findings. This presentation often complicates the diagnostic process; however, differential diagnosis can be achieved via a combination of clinical symptoms and rapid culture sampling.

Mycobacterium tuberculosis complex detection in rural goat herds in South Africa using Bayesian latent class analysis

Animal tuberculosis affects a wide range of domestic and wild animal species, including goats (Capra hircus). In South Africa, Mycobacterium tuberculosis complex (MTBC) testing and surveillance in domestic goats is not widely applied, potentially leading to under recognition of goats as a potential source of M. bovis spread to cattle as well as humans and wildlife. The aim of this study was to estimate diagnostic test performance for four assays and determine whether M. bovis infection was present in goats sharing communal pastures with M. bovis positive cattle in the Umkhanyakude district of Northern Zululand, KwaZulu Natal. In 2019, 137 M. bovis-exposed goats were screened for MTBC infection with four diagnostic tests: the in vivo single intradermal comparative cervical tuberculin test (SICCT), in vitro QuantiFERON®-TB Gold (QFT) bovine interferon-gamma release assay (IGRA), QFT bovine interferon gamma induced protein 10 (IP-10) release assay (IPRA), and nasal swabs tested with the Cepheid GeneXpert® MTB/RIF Ultra (GXU) assay for detection of MTBC DNA. A Bayesian latent class analysis was used to estimate MTBC prevalence and diagnostic test sensitivity and specificity. Among the 137 M. bovis-exposed goats, positive test results were identified in 15/136 (11.0%) goats by the SICCT; 4/128 (3.1%) goats by the IPRA; 2/128 (1.6%) goats by the IGRA; and 26/134 (19.4%) nasal swabs by the GXU. True prevalence was estimated by our model to be 1.1%, suggesting that goats in these communal herds are infected with MTBC at a low level. Estimated posterior means across the four evaluated assays ranged from 62.7% to 80.9% for diagnostic sensitivity and from 82.9% to 97.9% for diagnostic specificity, albeit estimates of the former (diagnostic sensitivity) were dependent on model assumptions. The application of a Bayesian latent class analysis and multiple ante-mortem test results may improve detection of MTBC, especially when prevalence is low. Our results provide a foundation for further investigation to confirm infection in communal goat herds and identify previously unrecognized sources of intra- and inter-species transmission of MTBC.

Culture-Independent PCR Detection and Differentiation of Mycobacteria spp. in Antemortem Respiratory Samples from African Elephants (Loxodonta Africana) and Rhinoceros (Ceratotherium Simum, Diceros Bicornis) in South Africa.

Since certain Mycobacterium tuberculosis complex (MTBC) members, such as M. bovis, are endemic in specific South African wildlife reserves and zoos, cases of clinically important nontuberculous mycobacteria (NTM) in wildlife may be neglected. Additionally, due to the inability of tests to differentiate between the host responses to MTBC and NTM, the diagnosis of MTBC may be confounded by the presence of NTMs. This may hinder control efforts. These constraints highlight the need for enhanced rapid detection and differentiation methods for MTBC and NTM, especially in high MTBC burden areas. We evaluated the use of the GeneXpert MTB/RIF Ultra, the Hain CMdirect V1.0 line probe assay, and novel amplicon sequencing PCRs targeting the mycobacterial rpoB and ku gene targets, directly on antemortem African elephant (n = 26) bronchoalveolar lavage fluid (BALF) (n = 22) and trunk washes (n = 21) and rhinoceros (n = 23) BALF (n = 23), with known MTBC culture-positive and NTM culture-positive results. Our findings suggest that the Ultra is the most sensitive diagnostic test for MTBC DNA detection directly in raw antemortem respiratory specimens and that the rpoB PCR is ideal for Mycobacterium genus DNA detection and species identification through amplicon sequencing.

Detection of Mycobacterium tuberculosis complex DNA in oronasal swabs from infected African buffaloes (Syncerus caffer)

Mycobacterium bovis (M. bovis), a member of the Mycobacterium tuberculosis complex (MTBC), is the causative agent of bovine TB (bTB) in animals. Spread occurs through inhalation or ingestion of bacilli transmitted from infected individuals. Early and accurate detection of infected African buffaloes shedding M. bovis is essential for interrupting transmission. In this pilot study, we determined if MTBC DNA could be detected in M. bovis infected buffalo oronasal secretions using a molecular transport media (PrimeStore MTM) with oronasal swabs and a rapid qPCR assay (Xpert MTB/RIF Ultra). Bovine TB test-positive buffaloes were culled, then tissue samples and oronasal swabs collected post-mortem for mycobacterial culture and Ultra testing, respectively. The Ultra detected MTBC DNA in 5/12 swabs from M. bovis culture-confirmed buffaloes. Oronasal swabs from M. bovis negative buffaloes (n = 20) were negative on Ultra, indicating the high specificity of this test. This study showed that MTM can successfully preserve MTBC DNA in oronasal swabs. The proportion of MTBC positive oronasal swabs was higher than expected and suggests that the Ultra may be an additional method for identifying infected buffaloes. Further studies are needed to confirm the utility of the Ultra assay with oronasal swabs as an assay to evaluate possible MTBC shedding in buffaloes.

Sternal swelling presenting as tuberculosis: a case report

BackgroundTuberculosis continues to be a worldwide public health problem. Despite the noted gradual decline in tuberculosis case rates in the UK, clinicians should still be aware of these unusual presentations. Sternal tuberculosis is an uncommon form of extrapulmonary tuberculosis and it can initially be a diagnostic challenge for paediatricians. These lesions can present with nonspecific signs and symptoms that may mimic malignancy.Case presentationWe present a case of a 3-year-old African descent girl with a sternal swelling that was confirmed to be Mycobacterium tuberculosis complex DNA on gastric aspirate. The child had additional radiological investigations that corresponded accordingly. She was started on quadruple antituberculosis therapy with good outcome.ConclusionTuberculosis sternal abscess is as rare finding, especially in developed countries where tuberculosis is not endemic. Tuberculosis may not always present with pulmonary symptoms in children. There should be a high suspicion of tuberculosis, especially in immigrant population presenting with unusual presentations. Our aim is to increase awareness around atypical presentations of tuberculosis in children. Although, tuberculosis is endemic to underdeveloped countries, clinicians should still be aware of presentations in view of current global migration.

A Comprehensive Evaluation of GeneLEAD VIII DNA Platform Combined to Deeplex Myc-TB® Assay to Detect in 8 Days Drug Resistance to 13 Antituberculous Drugs and Transmission of Mycobacterium tuberculosis Complex Directly From Clinical Samples

The GeneLEAD VIII (Diagenode, Belgium) is a new, fully automated, sample-to-result precision instrument for the extraction of DNA and PCR detection of Mycobacterium tuberculosis complex (MTBC) directly from clinical samples. The Deeplex Myc-TB® assay (Genoscreen, France) is a diagnostic kit based on the deep sequencing of a 24-plexed amplicon mix allowing simultaneously the detection of resistance to 13 antituberculous (antiTB) drugs and the determination of spoligotype. We evaluated the performance of a strategy combining the both mentioned tools to detect directly from clinical samples, in 8 days, MTBC and its resistance to 13 antiTB drugs, and identify potential transmission of strains from patient-to-patient. Using this approach, we screened 112 clinical samples (65 smear-negative) and 94 MTBC cultured strains. The sensitivity and the specificity of the GeneLEAD/Deeplex Myc-TB approach for MTBC detection were 79.3% and 100%, respectively. One hundred forty successful Deeplex Myc-TB results were obtained for 46 clinical samples and 94 strains, a total of 85.4% of which had a Deeplex Myc-TB susceptibility and resistance prediction consistent with phenotypic drug susceptibility testing (DST). Importantly, the Deeplex Myc-TB assay was able to detect 100% of the multidrug-resistant (MDR) MTBC tested. The lowest concordance rates were for pyrazinamide, ethambutol, streptomycin, and ethionamide (84.5%, 81.5%, 73%, and 55%, respectively) for which the determination of susceptibility or resistance is generally difficult with current tools. One of the main difficulties of Deeplex Myc-TB is to interpret the non-synonymous uncharacterized variants that can represent up to 30% of the detected single nucleotide variants. We observed a good level of concordance between Deeplex Myc-TB-spoligotyping and MIRU-VNTR despite a lower discriminatory power for spoligotyping. The median time to obtain complete results from clinical samples was 8 days (IQR 7–13) provided a high-throughput NGS sequencing platform was available. Our results highlight that the GeneLEAD/Deeplex Myc-TB approach could be a breakthrough in rapid diagnosis of MDR TB in routine practice.

Shared use of mineral supplement in extensive farming and its potential for infection transmission at the wildlife-livestock interface

Recently, the survival of Mycobacterium bovis on livestock mineral blocks has been confirmed, but little is known about its implication in the transmission of animal tuberculosis (TB) under field conditions. The objective of this study was to describe the shared use of mineral supplements in four extensive beef cattle farms from a high TB prevalence area in South Central Spain, to identify the main factors explaining their use, and characterize its potential role for the transmission of Mycobacterium tuberculosis Complex (MTC). This is relevant to design control measures at the wildlife-livestock interface. Animal activity was monitored by camera-trapping at 12 mineral supplementation points during spring and fall. Additionally, swabs were periodically taken from the mineral substrates and analyzed by PCR searching for MTC DNA. Cattle, pig, goat, sheep, wild boar, and red deer were all recorded licking on mineral supplementation points. Livestock species were the main users and presented a diurnal use pattern. Wild ungulates presented a nocturnal-crepuscular use pattern, with scarce overlapping with livestock. Wild boar presence was positively related to cattle presence at mineral supplementation points, whereas red deer presence was higher in supplemental points closer to forested areas and in farms without hunting pressure. We recorded 266 indirect wildlife-livestock interactions (i.e., two consecutive visits that occurred within 78 h), all of them derived from 21 unique wildlife visits. All the analyzed swabs resulted negative to MTC DNA. Comparing to other environmental sources of MTC in our study area, mainly water ponds, this research evidenced that mineral blocks are less attractive to wildlife. However, the potential for interspecific transmission of MTC or other pathogens cannot be discarded. The risk for interaction at mineral supplementation points and further transmission can be prevented by implementing specific measures in the context of integral biosecurity plans at the wildlife-livestock interface, which are proposed.

Performance of the Abbott RealTime MTB and RIF/INH resistance assays for the detection of Mycobacterium Tuberculosis and resistance markers in sputum specimens.

BackgroundThe Abbott RealTime MTB is an assay for the detection of Mycobacterium tuberculosis (MTB) complex DNA from respiratory specimens in combination with the Abbott RealTime RIF/INH assay for the detection of genetic resistance markers for isoniazid (INH) and rifampicin (RIF) from MTB positive isolates. Hence, this study aimed to evaluate the performance of the Abbott RealTime MTB and RIF/INH assays.MethodsA cross-sectional study was conducted on 289 study subjects presumptive to have pulmonary tuberculosis at Nigist Eleni Mohammed Memorial Hospital, South Ethiopia from April 2017 to June 2018. Two morning expectorated sputum specimens were collected from each study participant. One sample was tested directly by Xpert MTB/RIF assay at Nigist Eleni Mohammed Memorial Hospital and the other sample was used for smear microscopy, TB culture, Abbott RealTime MTB, and Abbott RealTime INH/RIF assays at International Clinical Laboratories, Addis Ababa, Ethiopia. The diagnostic performance of the Abbott RealTime MTB and INH/RIF assays were calculated against MGIT liquid culture and phenotypic drug susceptibility testing (DST) as the gold standard.ResultsFor the detection of MTB the Abbott RealTime MTB assay exhibited sensitivity 92.4% (95% CI 83.6–96.9), specificity 95.4% (95% CI 91.1–97.7), PPV 89.0% (95% CI 79.7–94.5) and NPV 96.9% (95% CI 93.0–98.7). For the detection of RIF resistance MTB, Abbott RealTime MTB RIF/INH concurred with phenotypic DST and Xpert MTB/RIF, while for the detection of INH resistance MTB, the sensitivity, specificity, PPV and NPV of the Abbott MTB RIF/INH assay was 84.2% (95% CI 60.4–96.6), 100% (95% CI 89.7–100), 100% and 91.9% (95% CI 80.0–96.9), respectively.ConclusionsThe Abbott RTMTB and RIF/INH assays revealed high sensitivity and specificity in MTB diagnosis and provided reliable INH and RIF resistance profiles. This assay has a similar diagnostic performance to the Xpert MTB/RIF assay with the advantages of high-throughput.

Diagnostic accuracy of the FluoroType MTB and MTBDR VER 2.0 assays for the centralized high-throughput detection of Mycobacterium tuberculosis complex DNA and isoniazid and rifampicin resistance

ObjectivesTo evaluate the accuracy of two new molecular diagnostic tests for the detection of drug-resistant tuberculosis, the FluoroType MTB and MTBDR VER 2.0 assays, in combination with manual and automated DNA extraction methods. MethodsSputa from 360 Xpert Ultra Mycobacterium tuberculosis complex (MTBC)-positive patients and 250 Xpert Ultra MTBC-negative patients were tested. GenoType MTBDRplus served as reference for MTBC and drug resistance detection. Sanger sequencing was used to resolve discrepancies. ResultsFluoroType MTB VER 2.0 showed similar MTBC sensitivity compared with FluoroType MTBDR VER 2.0 (manual DNA extraction: 91.6% (294/321) versus 89.8% (291/324); p 0.4); automated DNA extraction: 92.1% (305/331) versus 87.7% (291/332); p 0.05)). FluoroType MTBDR VER2.0 showed comparable diagnostic accuracy to FluoroType MTBDR VER1.0 as previously reported for the detection of MTBC and rifampicin and isoniazid resistance. ConclusionsThe FluoroType MTB and MTBDR VER 2.0 assays together with an automated DNA extraction and PCR set-up platform may improve laboratory operational efficiency for the diagnosis of MTBC and resistance to rifampicin and isoniazid and show promise for the implementation in a centralized molecular drug susceptibility testing model.