Diagnostic potential of combining plasma biomarkers of tissue damage and inflammation in pediatric TB.

INTRODUCTION: Immune-based diagnostic tests for tuberculosis (TB) have suboptimal sensitivity in children and cannot differentiate between latent infection (LTBI) and active disease. This study evaluated the diagnostic potential of a broad range of biomarkers of tissue damage and inflammation in unstimulated plasma in children. METHODS: We analyzed 17 biomarkers in 15 non-M. tuberculosis (MTB)-infected controls and 33 children with TB infection (LTBI, n = 8; probable TB, n = 19; confirmed TB, n = 6). Biomarker concentrations were measured using a Luminex magnetic bead-based platform and multiplex sandwich immunoassays. Concentrations, correlations and diagnostic accuracy assessments were conducted among patient groups. RESULTS: Confirmed TB cases had significantly higher concentrations of IFN-γ and IL-2 and higher IFN-γ/MCP-1 and IL-2/MCP-1 ratios compared to LTBI and non-MTB-infected children. Among children with confirmed TB, there was a strong correlation between IFN-γ and IL-10 (r = 0.95; p < 0.001) and a significant correlation between IL-2 and IL-1ra (r = 0.92), IL-21 (r = 0.91), MCP-3 (r = 0.84), and MMP-1 (r = 0.85). The IFN-γ/MCP-1 ratio was the most accurate biomarker combination for differentiating between MTB-infected and non-MTB-infected children (AUC, 0.82; sensitivity, 87.9%; specificity, 66.6%; p < 0.001) and between active TB and non-MTB-infected children (AUC 0.82; sensitivity 88.0%; specificity 60.0%; p < 0.001). None of the biomarkers investigated were able to discriminate between LTBI and active TB. CONCLUSION: Our data suggest that combining the analyses of multiple biomarkers in plasma has the potential to enhance diagnosis of TB in children and, thus, warrants additional investigation. In particular, the diagnostic potential of IFN-γ/MCP-1 ratios should be further explored in larger pediatric cohorts.

QuantiFERON-TB reversion in children and adolescents with tuberculosis.

We analyzed 136 children with tuberculosis disease or infection and a positive QuantiFERON-TB (QFT) assay, followed-up for a median of 21 months (0.4-11years). QFT reversed in 16.9% of cases, with significant decreases in TB1 (-1.72 vs. -0.03 IU/ml, p=0.001) and TB2 (-1.65 vs. -0.43 IU/ml, p=0.005) levels compared to non-reverters. We found a higher QFT reversion rate among children under 5 years (25.0% vs 11.9%, p=0.042), and those with TST induration <15mm (29% vs 13.3%, p=0.055). Our data reveal that, although QFT test remained positive in the majority of children, reversion occurred in 16% of cases in a progressive and stable pattern. Younger age and reduced TST induration were associated with QFT reversion.

Microevolution, reinfection and highly complex genomic diversity in patients with sequential isolates of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic, extensively drug-resistant non-tuberculous mycobacterium. Few genomic studies consider its diversity in persistent infections. Our aim was to characterize microevolution/reinfection events in persistent infections. Fifty-three sequential isolates from 14 patients were sequenced to determine SNV-based distances, assign resistance mutations and characterize plasmids. Genomic analysis revealed 12 persistent cases (0-13 differential SNVs), one reinfection (15,956 SNVs) and one very complex case (23 sequential isolates over 192 months), in which a first period of persistence (58 months) involving the same genotype 1 was followed by identification of a genotype 2 (76 SNVs) in 6 additional alternating isolates; additionally, ten transient genotypes (88-243 SNVs) were found. A macrolide resistance mutation was identified from the second isolate. Despite high diversity, the genotypes shared a common phylogenetic ancestor and some coexisted in the same specimens. Genomic analysis is required to access the true intra-patient complexity behind persistent infections involving M. abscessus.

Pain assessment and management among adult patients in a gynecology ward: a best practice implementation project.

OBJECTIVES: This project aimed to improve compliance with evidence-based practice in pain assessment and management in a gynecology ward. INTRODUCTION: Effective pain control is important to prevent the negative consequences of pain that is poorly managed. However, it remains undervalued and inadequately treated. Applying evidence-based practices to correctly evaluate and manage pain is essential to improve pain relief. METHODS: This project followed the JBI Evidence Implementation Framework. A baseline audit of 41 women admitted to the gynecology ward was conducted and measured against 5 best practice criteria, along with a patient satisfaction questionnaire. Targeted strategies were then implemented and a follow-up audit was conducted using the same criteria, methods, and sample size as the baseline audit. RESULTS: The baseline audit revealed gaps between current and best practice. Barriers to implementation were identified and strategies to resolve the barriers were designed and implemented (nurse education, informative materials, electronic patient records system improvements). Comprehensive pain assessment, including dynamic and static pain assessment, use of a validated tool, and education provided to patients and carers, improved in the follow-up audit. There was no change in patient satisfaction levels; however, the discrepancy between pain measured by nurses and pain measured by patients was reduced. CONCLUSIONS: The JBI methodology was useful in improving compliance with evidence-based practice criteria. It also facilitated adaptation to new barriers, such as the COVID-19 pandemic. Improving nurses' knowledge of pain assessment can lead to more accurate assessment. Inadequate records systems also made it difficult to record the care that was provided. Subsequent audits will assess sustainability and the project will be escalated to other wards.

Development and characterization of a bacteriophage cocktail with high lytic efficacy against field-isolated Salmonella enterica.

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

Subspecies Distribution and Antimicrobial Susceptibility Testing of Mycobacterium abscessus Clinical Isolates in Madrid, Spain: a Retrospective Multicenter Study.

Mycobacterium abscessus (MABS) is the most pathogenic and drug-resistant rapidly growing mycobacteria. However, studies on MABS epidemiology, especially those focusing on subspecies level, are scarce. We aimed to determine MABS subspecies distribution and its correlation with phenotypic and genotypic antibiotic profiles. A retrospective multicenter study of 96 clinical MABS isolates in Madrid between 2016 to 2021 was conducted. Identification at the subspecies level and resistance to macrolides and aminoglycosides were performed by the GenoType NTM-DR assay. The MICs of 11 antimicrobials tested against MABS isolates were determined using the broth microdilution method (RAPMYCOI Sensititer titration plates). Clinical isolates included 50 (52.1%) MABS subsp. abscessus; 33 (34.4%) MABS subsp. massiliense; and 13 (13.5%) MABS subsp. bolletii. The lowest resistance rates corresponded to amikacin (2.1%), linezolid (6.3%), cefoxitin (7.3%), and imipenem (14.6%), and the highest to doxycycline (100.0%), ciprofloxacin (89.6%), moxifloxacin (82.3%), cotrimoxazole (82.3%), tobramycin (81.3%), and clarithromycin (50.0% at day 14 of incubation). Regarding tigecycline, although there are no susceptibility breakpoints, all strains but one showed MICs ≤ 1 µg/mL. Four isolates harbored mutations at positions 2058/9 of the rrl gene, one strain harbored a mutation at position 1408 of the rrl gene, and 18/50 harbored the T28C substitution at erm(41) gene. Agreement of the GenoType results with clarithromycin and amikacin susceptibility testing was 99.0% (95/96). The rate of MABS isolates showed an upward trend during the study period, being M. abscessus subsp. abscessus the most frequently isolated subspecies. Amikacin, cefoxitin, linezolid, and imipenem showed great in vitro activity. The GenoType NTM-DR assay provides a reliable and complementary tool to broth microdilution for drug resistance detection. IMPORTANCE Infections caused by Mycobacterium abscessus (MABS) are increasingly being reported worldwide. Identifying MABS subspecies and assessing their phenotypic resistance profiles are crucial for optimal management and better patient outcomes. M. abscessus subspecies differ in erm(41) gene functionality, which is a critical determinant of macrolide resistance. Additionally, resistance profiles of MABS and the subspecies distribution can vary geographically, highlighting the importance of understanding local epidemiology and resistance patterns. This study provides valuable insights into the epidemiology and resistance patterns of MABS and its subspecies in Madrid. Elevated resistance rates were observed for several recommended antimicrobials, emphasizing the need for cautious drug use. Furthermore, we assessed the GenoType NTM-DR assay, which examines principal mutations in macrolides and aminoglycosides resistance-related genes. We observed a high level of agreement between the GenoType NTM-DR assay and the microdilution method, indicating its usefulness as an initial tool for early initiation of appropriate therapy.

Clinical Evaluation of Nontuberculous Mycobacteria (NTM) Elite Agar, a New Medium for the Isolation of NTM: a Multicenter Study.

Nontuberculous mycobacteria (NTM) are gaining interest with the increased number of infected patients. NTM Elite agar is designed specifically for the isolation of NTM without the decontamination step. We assessed the clinical performance of this medium combined with Vitek mass spectrometry (MS) matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology for the isolation and identification of NTM in a prospective multicenter study, including 15 laboratories (24 hospitals). A total of 2,567 samples from patients with suspected NTM infection were analyzed (1,782 sputa, 434 bronchial aspirates, 200 bronchoalveolar lavage samples, 34 bronchial lavage samples, and 117 other samples). A total of 220 samples (8.6%) were positive with existing laboratory methods against 330 with NTM Elite agar (12.8%). Using the combination of both methods, 437 isolates of NTM were detected in 400 positive samples (15.6% of samples). In total, 140 samples of the standard procedures (SP) and 98 of the NTM Elite agar were contaminated. NTM Elite agar showed a higher performance for rapidly growing mycobacteria (RGM) species than SP (7% versus 3%, P < 0.001). A trend has been noted for the Mycobacterium avium complex (4% with SP versus 3% with NTM Elite agar, P = 0.06). The time to positivity was similar (P = 0.13) between groups. However, the time to positivity was significantly shorter for the RGM in subgroup analysis (7 days with NTM and 6 days with SP, P = 0.01). NTM Elite agar has been shown to be useful for the recovery of NTM species, especially for the RGM. Using NTM Elite agar + Vitek MS system in combination with SP increases the number of NTM isolated from clinical samples.

Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning.

Mycobacterium abscessus is one of the most common and pathogenic nontuberculous mycobacteria (NTM) isolated in clinical laboratories. It consists of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Due to their different antibiotic susceptibility pattern, a rapid and accurate identification method is necessary for their differentiation. Although matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has proven useful for NTM identification, the differentiation of M. abscessus subspecies is challenging. In this study, a collection of 325 clinical isolates of M. abscessus was used for MALDI-TOF MS analysis and for the development of machine learning predictive models based on MALDI-TOF MS protein spectra. Overall, using a random forest model with several confidence criteria (samples by triplicate and similarity values >60%), a total of 96.5% of isolates were correctly identified at the subspecies level. Moreover, an improved model with Spanish isolates was able to identify 88.9% of strains collected in other countries. In addition, differences in culture media, colony morphology, and geographic origin of the strains were evaluated, showing that the latter had an impact on the protein spectra. Finally, after studying all protein peaks previously reported for this species, two novel peaks with potential for subspecies differentiation were found. Therefore, machine learning methodology has proven to be a promising approach for rapid and accurate identification of subspecies of M. abscessus using MALDI-TOF MS.

Identification of Nocardia and non-tuberculous Mycobacterium species by MALDI-TOF MS using the VITEK MS coupled to IVD and RUO databases.

Identification of Nocardia and Mycobacterium species by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is still a challenging task that requires both suitable protein extraction procedures and extensive databases. This study aimed to evaluate the VITEK MS Plus system coupled with updated RUO (v4.17) and IVD (v3.2) databases for the identification of Nocardia spp. and Mycobacterium spp. clinical isolates. Sample preparation was carried out using the VITEK MS Mycobacterium/Nocardia kit for protein extraction. From 90 Nocardia spp. isolates analysed, 86 (95.6%) were correctly identified at species or complex level using IVD and 78 (86.7%) using RUO. Only two strains were misidentified as other species pertaining to the same complex. Among the 106 non-tuberculous Mycobacterium clinical isolates tested from a liquid culture medium, VITEK MS identified correctly at species or complex level 96 (90.6%) isolates in the IVD mode and 89 (84.0%) isolates in the RUO mode. No misidentifications were detected. Although the IVD mode was unable to differentiate members of the M. fortuitum complex, the RUO mode correctly discriminated M. peregrinum and M. septicum. The robustness and accuracy showed by this system allow its implementation for routine identification of these microorganisms in clinical laboratories.

Accuracy of Xpert Ultra for the diagnosis of paediatric tuberculosis in a low TB burden country: a prospective multicentre study.

INTRODUCTION: Childhood pulmonary tuberculosis (TB) remains a diagnostic challenge. This study aimed to evaluate the performance of Xpert Ultra for the diagnosis of pulmonary TB in children in a low TB prevalence setting. METHODS: Prospective, multicentre, diagnostic accuracy study. Children with clinical or radiological suspicion of pulmonary TB were recruited at 11 paediatric units in Spain. Up to three gastric or sputum specimens were taken on 3 consecutive days, and analysed by Xpert MTB/RIF, Xpert Ultra and culture in parallel. RESULTS: 86 children were included (median age 4.9 years, IQR 2.0-10.0; 51.2% male). The final diagnosis was pulmonary TB in 75 patients (87.2%); 33 (44.0%) were microbiologically confirmed. A total of 219 specimens, comprising gastric aspirates (n=194; 88.6%) and sputum specimens (n=25; 11.4%), were analysed. Using culture as reference standard and comparing individual specimens, the sensitivity was 37.8% (14/37) for Xpert MTB/RIF and 81.1% (30/37) for Xpert Ultra (p<0.001); specificity was 98.4% (179/182) and 93.4% (170/182), respectively (p=0.02). In the per-patient analysis, considering positive results on any specimen, the sensitivity was 42.9% (9/21) for Xpert MTB/RIF and 81.0% for Xpert Ultra (17/21, p=0.01); specificity was 96.9% (63/65) and 87.7% (57/65, p=0.07), respectively. CONCLUSIONS: In children with pulmonary TB in a low burden setting, Xpert Ultra has significantly higher sensitivity than the previous generation of Xpert assay and only marginally lower specificity. Therefore, in children undergoing evaluation for suspected pulmonary TB, Xpert Ultra should be used in preference to Xpert MTB/RIF whenever possible.

Long-Term Interactions of Salmonella Enteritidis With a Lytic Phage for 21 Days in High Nutrients Media.

Salmonella spp. is a relevant foodborne pathogen with worldwide distribution. To mitigate Salmonella infections, bacteriophages represent an alternative to antimicrobials and chemicals in food animals and food in general. Bacteriophages (phages) are viruses that infect bacteria, which interact constantly with their host. Importantly, the study of these interactions is crucial for the use of phages as a mitigation strategy. In this study, experimental coevolution of Salmonella Enteritidis (S. Enteritidis) and a lytic phage was conducted in tryptic soy broth for 21 days. Transfer to fresh media was conducted daily and every 24 hours, 2 mL of the sample was collected to quantify Salmonella OD600 and phage titter. Additionally, time-shift experiments were conducted on 20 colonies selected on days 1, 12, and 21 to evaluate the evolution of resistance to past (day 1), present (day 12), and future (day 21) phage populations. The behavior of the dynamics was modeled and simulated with mathematical mass-action models. Bacteria and phage from days 1 and 21 were sequenced to determine the emergence of mutations. We found that S. Enteritidis grew for 21 days in the presence and absence of the phage and developed resistance to the phage from day 1. Also, the phage was also able to survive in the media for 21 days, however, the phage titer decreased in approx. 3 logs PFU/mL. The stability of the lytic phage population was consistent with the leaky resistance model. The time-shift experiments showed resistance to phages from day 1 of at least 85% to the past, present, and future phages. Sequencing of S. Enteritidis showed mutations in genes involved in lipopolysaccharide biosynthesis genes rfbP and rfbN at day 21. The phage showed mutations in the tail phage proteins responsible for recognizing the cell surface receptors. These results suggest that interactions between bacteria and phage in a rich resource media generate a rapid resistance to the infective phage but a fraction of the population remains susceptible. Interactions between Salmonella and lytic phages are an important component for the rational use of phages to control this important foodborne pathogen.

Effect of Intramuscularly Administered Oxytetracycline or Enrofloxacin on Vancomycin-Resistant Enterococci, Extended Spectrum Beta-Lactamase- and Carbapenemase-Producing Enterobacteriaceae in Pigs.

Nowadays, there is a great concern about the prevalence of multidrug resistant Enterococcus spp. and Enterobacteriaceae in food-producing animals. The aim of this work was to evaluate the effect of oxytetracycline or enrofloxacin treatment on vancomycin-resistant enterococci (VRE), extended spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae in pigs. A total of 26 piglets were received and distributed in three groups. Group 1 was treated with enrofloxacin (N = 12), group 2 with oxytetracycline (N = 10) and group 3 did not receive any treatment (control group) (N = 4). A higher number of vancomycin-resistant E. faecium were recovered compared to E. faecalis. In the pigs treated with enrofloxacin, vancomycin resistant E. faecium was found in a higher percentage of animals than in the control group. ESBL-producing E. coli was not detected in rectal samples from control animals. However, it was detected in 17-20% of animals treated with oxytetracycline on days 6 to 17 and in 17-50% of the animals treated with enrofloxacin. Carbapenemase-producing E. coli was isolated in animals treated with oxytetracycline, but not in animals treated with enrofloxacin or in the control group. This study highlights that the use of oxytetracycline or enrofloxacin in food-producing animals could select ESBL and carbapenemase-producing E. coli. Further studies shall be needed to validate the results obtained, considering a more robust and extended experimental design.

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification.

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

Performance of QuantiFERON-TB Gold Plus assays in children and adolescents at risk of tuberculosis: a cross-sectional multicentre study.

INTRODUCTION: The QuantiFERON-TB Gold Plus (QFT-Plus) assay, which features two antigen-stimulated tubes (TB1 and TB2) instead of a single tube used in previous-generation interferon-gamma release assays (IGRAs), was launched in 2016. Despite this, data regarding the assay's performance in the paediatric setting remain scarce. This study aimed to determine the performance of QFT-Plus in a large cohort of children and adolescents at risk of tuberculosis (TB) in a low-burden setting. METHODS: Cross-sectional, multicentre study at healthcare institutions participating in the Spanish Paediatric TB Research Network, including patients <18 years who had a QFT-Plus performed between September 2016 and June 2020. RESULTS: Of 1726 patients (52.8% male, median age: 8.4 years), 260 (15.1%) underwent testing during contact tracing, 288 (16.7%) on clinical/radiological suspicion of tuberculosis disease (TBD), 649 (37.6%) during new-entrant migrant screening and 529 (30.6%) prior to initiation of immunosuppressive treatment. Overall, the sensitivity of QFT-Plus for TBD (n=189) and for latent tuberculosis infection (LTBI, n=195) was 83.6% and 68.2%, respectively. The agreement between QFT-Plus TB1 and TB2 antigen tubes was excellent (98.9%, κ=0.961). Only five (2.5%) patients with TBD had discordance between TB1 and TB2 results (TB1+/TB2-, n=2; TB1-/TB2+, n=3). Indeterminate assay results (n=54, 3.1%) were associated with young age, lymphopenia and elevated C reactive protein concentrations. CONCLUSIONS: Our non-comparative study indicates that QFT-Plus does not have greater sensitivity than previous-generation IGRAs in children in both TBD and LTBI. In TBD, the addition of the second antigen tube, TB2, does not enhance the assay's performance substantially.

Development of a new dual electrochemical immunosensor for a rapid and sensitive detection of enrofloxacin in meat samples.

A novel dual electrochemical immunosensor was fabricated for the rapid and sensitive detection of enrofloxacin (EF) antibiotic in meat. Anti-quinolone antibody was immobilized onto screen-printed dual carbon electrodes via carbodiimide coupling. A new electrochemical probe was synthesized by conjugating difloxacin and aminoferrocene, whose oxidation was measured at + 0.2 V vs. Ag/AgCl by differential pulse voltammetry. The detection principle was based on the competitive binding of this conjugate and free EF on immobilized antibodies. The proposed immunosensor allowed detection of EF at concentrations ranging from 0.005 µg.mL-1 to 0.01 µg.mL-1 with a detection limit of 0.003 µg.mL-1. The immunosensor was stable for at least 1 month at 4 °C and displayed a good specificity for other fluoroquinolones. The new dual electrode design offered an improved accuracy as one electrode was used as negative control. The efficiency of the sensor and the adequacy of the extraction process were finally validated by detecting EF in different meat samples.

Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy.

Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients' disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. IMPORTANCE We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences.

Optimization and Validation of a New Microbial Inhibition Test for the Detection of Antimicrobial Residues in Living Animals Intended for Human Consumption.

Even though antibiotics are necessary in livestock production, they can be harmful not only due to their toxicity, but also in view of their contribution to the emergence of antimicrobial resistance. Screening tests based on microbial growth inhibition appeared to be useful tools to prevent its entry into the food chain. They have nevertheless been traditionally carried out post mortem, leading to great economical loss and harm to the environment in case a positive sample is found. Hence, the objective was to evaluate the use of a screening test as an ante mortem alternative for the detection of antibiotic residues in meat: thus, Explorer®-Blood test was optimized and validated. After adapting the procedure for matrix preparation, the assay parameters were assessed from 344 antibiotic-free blood serum samples. Limits of Detection (LoDs) were defined by spiking blood serum with several of the most common antimicrobials used in veterinary practice. LoDs were similar to those obtained for meat and were at or below the maximum residue limits set by EU legislation for muscle. Analyses of in vivo injected samples, previously characterized by LC-MS/MS, demonstrated the method's accuracy and proved that Explorer®-Blood can be considered a suitable alternative to conventional post mortem screening methods.

Antibacterial Residue Excretion via Urine as an Indicator for Therapeutical Treatment Choice and Farm Waste Treatment.

Many of the infectious diseases that affect livestock have bacteria as etiological agents. Thus, therapy is based on antimicrobials that leave the animal's tissues mainly via urine, reaching the environment through slurry and waste water. Once there, antimicrobial residues may lead to antibacterial resistance as well as toxicity for plants, animals, or humans. Hence, the objective was to describe the rate of antimicrobial excretion in urine in order to select the most appropriate molecule while reducing harmful effects. Thus, 62 pigs were treated with sulfamethoxypyridazine, oxytetracycline, and enrofloxacin. Urine was collected through the withdrawal period and analysed via LC-MS/MS. Oxytetracycline had the slowest rate of degradation (a half-life time of 4.18 days) and the most extended elimination period in urine (over 2 months), followed by enrofloxacin (a half-life time of 1.48 days, total urine elimination in ca. 3 weeks) and sulfamethoxypyridazine (a half-life time of 0.49 days, total urine elimination in ca. 1 week). Bacterial sensitivity and recommendations for responsible use are limiting when selecting the treatment. Nevertheless, with similar effectiveness, sulfamethoxypyridazine would be the choice, as waste treatment would only need to be implemented for 1 week after treatment. Thus, more in-depth knowledge regarding antibacterial elimination would improve resource management, while protecting animals and consumers' health.

Performance of Xpert MTB/RIF Ultra assay on respiratory and extra-respiratory samples in a high-resource setting with a low tuberculosis prevalence.

The Xpert MTB/RIF assay is a molecular assay that has improved the detection of tuberculosis and rifampicin resistance. However, its sensitivity is limited in patients with paucibacillary disease. Xpert MTB/RIF Ultra has been developed to resolve this limitation. We compared the performance of Xpert Ultra with that of culture for detection of Mycobacterium tuberculosis and rifampicin resistance. We reviewed laboratory records for 848 respiratory and 419 extrarespiratory samples that were processed between April 2018 and October 2019. The sensitivity, specificity, negative predictive value, and positive predictive value of Xpert Ultra were 94.8%, 98%, 98.8%, and 91.3% for respiratory samples and 83.8%, 96.9%, 98.4% and 72.1% for nonrespiratory ones. We found 26 culture-negative/Ultra-positive samples. Most of them have low bacillary burden and more than half belonged to patients with history of tuberculosis. Xpert Ultra demonstrates excellent diagnostic accuracy for tuberculosis detection, including paucibacillary specimens. In patients with history of tuberculosis, PCR results should be interpreted carefully.