Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS.
ABSTRACT: Respiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2-71.0%) and 100% specificity (Sp) (100-100%) after only 6?hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0-76.1%) and specificity (Sp) of 100% (100-100%). In mixed culture samples, containing ?2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1% Se (38.8-75.5%) and 100% Sp (100-100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use.
Project description:Mycoplasma bovis is a leading cause of pneumonia in modern calf rearing. Fast identification is essential to ensure appropriate antimicrobial therapy. Therefore, the objective of this study was to develop a protocol to identify M. bovis from bronchoalveolar lavage fluid (BALf) with matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS and to determine the diagnostic accuracy in comparison with other techniques. BALf was obtained from 104 cattle, and the presence of M. bovis was determined in the following three ways: (i) rapid identification of M. bovis with MALDI-TOF MS (RIMM) (BALf was enriched and after 24, 48, and 72 h of incubation and was analyzed using MALDI-TOF MS), (ii) triplex real-time PCR for M. bovis, Mycoplasma bovirhinis, and Mycoplasma dispar, and (iii) 10-day incubation on selective-indicative agar. The diagnostic accuracy of the three tests was determined with Bayesian latent class modeling (BLCM). After 24 h of enrichment, M. bovis was identified with MALDI-TOF MS in 3 out of 104 BALf samples. After 48 and 72 h of enrichment, 32/104 and 38/100 samples, respectively, were M. bovis positive. Lipase-positive Mycoplasma-like colonies were seen in 28 of 104 samples. Real-time PCR resulted in 28/104 positive and 12/104 doubtful results for M. bovis The BLCM showed a sensitivity (Se) and specificity (Sp) of 86.6% (95% credible interval [CI], 69.4% to 97.6%) and 86.4% (CI, 76.1 to 93.8) for RIMM. For real-time PCR, Se was 94.8% (CI, 89.9 to 97.9) and Sp was 88.9% (CI, 78.0 to 97.4). For selective-indicative agar, Se and Sp were 70.5% (CI, 52.1 to 87.1) and 93.9% (CI, 85.9 to 98.4), respectively. These results suggest that rapid identification of M. bovis with MALDI-TOF MS after an enrichment procedure is a promising test for routine diagnostics in veterinary laboratories.
Project description:Quantitative bacterial culture of bronchoalveolar lavage fluids (BALF) is labor-intensive, and the delay involved in performing culture, definitive identification, and susceptibility testing often results in prolonged use of broad-spectrum antibiotics. The Unyvero lower respiratory tract (LRT) panel (Curetis, Holzgerlingen, Germany) allows the multiplexed rapid detection and identification of 20 potential etiologic agents of pneumonia within 5?h of collection. In addition, the assay includes detection of gene sequences that confer antimicrobial resistance. We retrospectively compared the performance of the molecular panel to routine quantitative bacterial culture methods on remnant BALF. Upon testing 175 BALF, we were able to analyze positive agreement of 181 targets from 129 samples, and 46 samples were negative. The positive percent agreement (PPA) among the microbial targets was 96.5%, and the negative percent agreement (NPA) was 99.6%. The targets with a PPA of <100% were Staphylococcus aureus (34/37 [91.9%]), Streptococcus pneumoniae (10/11 [90.9%]), and Enterobacter cloacae complex (2/4 [50%]). For the analyzable resistance targets, concordance with phenotypic susceptibility testing was 79% (14/18). This study found the Unyvero LRT panel largely concordant with culture results; however, no outcome or clinical impact studies were performed.
Project description:Mycoplasma bovis is associated with several clinical syndromes of cattle. Currently, limited information is available on the sensitivity (Se) and specificity (Sp) of serological assays used for the detection of M. bovis-specific antibodies. Consequently, it is difficult to critically evaluate the outcomes of studies that use these assays. Therefore, the current study used bovine sera sourced from M. bovis exposure studies from three countries to estimate the Se and Sp of two commercial M. bovis enzyme-linked immunosorbent assays (ELISA), BIO K302 and BIO K260, and Western blotting. Western blotting had the highest Se estimate of 74% (95% confidence interval (CI): 16-98%), compared to the BIO K302: 47% (95% CI: 10-87%) and BIO K260: 28% (95% CI: 1-92%). However, for Sp, the BIO K302: 96% (95% CI: 87-99%) and the BIO K260: 100% (95% CI: 93-100%) out-performed Western blotting: 88% (95% CI: 56-98%). Western blotting was the best assay for detecting seroconversion, correctly identifying 61% (95% CI: 29-86%) of exposed animals compared to 35% for BIO K302 (95% CI: 21-54%) and 8% for BIO K260 (95% CI: 0-87%). While none of the methods assessed had high Se and Sp, the availability of these estimates will aid in the interpretation of studies that use these assays. The results of this study highlight the difficulties encountered when using serology to detect exposure to M. bovis in cattle.
Project description:Cell envelope proteins from <i>Mycobacterium avium</i> subspecies <i>paratuberculosis</i> (MAP) that are antigenically distinct from closely related mycobacterial species are potentially useful for Johne's Disease (JD) diagnosis. We evaluated the potential of ELISAs, based on six antigenically distinct recombinant MAP cell envelope proteins (SdhA, FadE25_2, FadE3_2, Mkl, DesA2, and hypothetical protein MAP1233) as well as an extract of MAP total cell envelope proteins, to detect antibodies against MAP in the sera of infected cattle. The sensitivity (Se) and specificity (Sp) of an ELISA based on MAP total cell envelope proteins, when analyzing 153 bovine serum samples, was 75 and 96%, respectively. Analysis of the same samples, using a commercial serum ELISA resulted in a Se of 56% and Sp of 99%. Results of ELISA analysis using plates coated with recombinant cell envelope proteins ranged from a highest Se of 94% and a lowest Sp of 79% for Sdh A to a lowest Se of 67% and a highest Sp of 95% for hypothetical protein MAP1233. Using polyclonal antibodies to MAP total cell envelope proteins, immunohistochemical analysis of intestinal and lymph node tissues from JD-positive cattle detected MAP organisms whereas antibodies to recombinant proteins did not. Finally, polyclonal antibodies to MAP total cell envelope protein and to recombinant SdhA, FadE25_2, and DesA2 proteins immunomagnetically separated MAP microorganisms spiked in PBS. These results suggest that antigenically distinct MAP cell envelope proteins and antibodies to these proteins may have potential to detect MAP infection in dairy cattle.
Project description:Surfactant protein B (SP-B) proprotein contains three saposin-like protein (SAPLIP) domains: a SAPLIP domain corresponding to the mature SP-B peptide is essential for lung function and postnatal survival; the function of SAPLIP domains in the N-terminal (SP-BN) and C-terminal regions of the proprotein is not known. In the current study, SP-BN was detected in the supernatant of mouse bronchoalveolar lavage fluid (BALF) and in nonciliated bronchiolar cells, alveolar type II epithelial cells, and alveolar macrophages. rSP-BN indirectly promoted the uptake of bacteria by macrophage cell lines and directly killed bacteria at acidic pH, consistent with a lysosomal, antimicrobial function. Native SP-BN isolated from BALF also killed bacteria but only at acidic pH; the bactericidal activity of BALF at acidic pH was completely blocked by SP-BN Ab. Transgenic mice overexpressing SP-BN and mature SP-B peptide had significantly decreased bacterial burden and increased survival following intranasal inoculation with bacteria. These findings support the hypothesis that SP-BN contributes to innate host defense of the lung by supplementing the nonoxidant antimicrobial defenses of alveolar macrophages.
Project description:<h4>Background</h4>The use of multiplex PCR to shorten time to identification of pathogens and their resistance mechanisms for patients with ventilator-associated pneumonia (VAP) is attractive, but poorly studied. The multiplex PCR-based Unyvero pneumonia cartridge assay can directly identify 20 bacteria and one fungus, amongst the most frequently causing VAP, and 19 of their resistance markers in clinical specimens (bronchoalveolar lavage or tracheal aspirate), with a turnaround time of 4-5 h. We performed this study to evaluate the concordance between the multiplex PCR-based Unyvero pneumonia cartridge assay and conventional microbiological techniques to identify pathogens and their resistance mechanisms in patients with VAP.<h4>Methods</h4>All patients suspected of having VAP (January 2016 to January 2019), who underwent fiberoptic bronchoscopy with bronchoalveolar lavage fluid (BALF) and whose BALF microscopy examination revealed intracellular bacteria, were included. BALF conventional cultures (gold standard), antimicrobial susceptibility testing and processing for the Unyvero pneumonia cartridge were done. Culture and Unyvero results were compared.<h4>Results</h4>Compared to cultures of the 93 samples processed for both techniques, Unyvero correctly identified pathogens in 68 (73%) proven VAP episodes, was discordant for 25 (27%), detected no pathogen in 11 and overdetected a not otherwise found pathogen in six. For the eight remaining discordant results, the pathogen responsible for VAP was not included in the Unyvero cartridge panel or it grew at a non-significant level in culture. Amongst the 31 (33%) resistance mechanism discordances observed, 22 were resistance detection failures and 24 concerned Pseudomonas aeruginosa.<h4>Conclusions</h4>Compared to conventional microbiological cultures, the Unyvero pneumonia cartridge had poor diagnostic performance: it correctly identified pathogens and their resistance mechanisms in 73% and 67% of VAP cases, respectively. The lack of performance on the resistance mechanism was more pronounced when the pathogen detected was a Pseudomonas aeruginosa.
Project description:The analysis of bronchoalveolar lavage fluid (BALF) using mass spectrometry-based metabolomics can provide insight into lung diseases, such as asthma. However, the important step of compound identification is hindered by the lack of a small molecule database that is specific for BALF. Here we describe prototypic, small molecule databases derived from human BALF samples (n=117). Human BALF was extracted into lipid and aqueous fractions and analyzed using liquid chromatography mass spectrometry. Following filtering to reduce contaminants and artifacts, the resulting BALF databases (BALF-DBs) contain 11,736 lipid and 658 aqueous compounds. Over 10% of these were found in 100% of samples. Testing the BALF-DBs using nested test sets produced a 99% match rate for lipids and 47% match rate for aqueous molecules. Searching an independent dataset resulted in 45% matching to the lipid BALF-DB compared to<25% when general databases are searched. The BALF-DBs are available for download from MetaboLights. Overall, the BALF-DBs can reduce false positives and improve confidence in compound identification compared to when general databases are used.
Project description:Infection with the Mycobacterium bovis (M. bovis) causes a disease referred to as bovine tuberculosis (bTB), which affects a wide range of mammal hosts. Many countries have implemented control and eradication plans that have resulted in variable levels of efficacy and success. Although bTB is a notifiable disease in Argentina, and a control plan that targets cattle herds has been in place for decades, M. bovis is still prevalent in cattle, swine, and certain wild species. The aim of the paper here was to assess the sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV) of PCR from tissue, which is a test for rapid M. bovis detection in swine. Bacteriological culture was also performed for comparison purposes. A Bayesian approach was applied to estimate the accuracy of the diagnostic tests, PCR and bacteriological culture, in 266 swine samples with bTB-like lesions recovered during routine official inspections at slaughterhouses. A one-population model, assuming conditional dependence between test results, and incorporating prior information on the performance of the tests obtained from the literature, was used to estimate the tests Se and Sp. The accuracy of the combined (in parallel) application of both tests was also estimated. The Se of the PCR (82.9%) was higher than the Se of the bacteriological culture (79.9%), whereas the Sp of both tests was similar (88.5 and 89.0%, respectively). Furthermore, when both techniques were assessed in parallel, the Se of the diagnostic system increased substantially (Se = 96.6%) with a moderate Sp loss (Sp = 78.8%; PPV = 92.8%; NPV = 89%). Results suggest that the PCR, or the combined application of bacteriological culture and PCR, may serve as an accurate diagnostic tool to confirm bTB in swine samples. Results here will help the design and implementation of effective surveillance strategies for the disease in swine of Argentina and other settings in which the disease is prevalent.
Project description:Rapid and low-cost pathogen diagnostic approaches are critical for clinical decision-making procedures. Cultivating bacteria often takes days to identify pathogens and provide antimicrobial susceptibilities. The delay in diagnosis may result in compromised treatment and inappropriate antibiotic use. Over the past decades, molecular-based techniques have significantly shortened pathogen identification turnaround time with high accuracy. However, these assays often use complex fluorescent labeling and nucleic acid amplification processes, which limit their use in resource-limited settings. In this work, we demonstrate a wash-free molecular agglutination assay with a straightforward mixing and incubation step that significantly simplifies procedures of molecular testing. By targeting the 16S rRNA gene of pathogens, we perform a rapid pathogen identification within 30 min on a dark-field imaging microfluidic cytometry platform. The dark-field images with low background noise can be obtained using a narrow beam scanning technique with off-the-shelf complementary metal oxide semiconductor (CMOS) imagers such as smartphone cameras. We utilize a machine learning algorithm to deconvolute topological features of agglutinated clusters and thus quantify the abundance of bacteria. Consequently, we unambiguously distinguish Escherichia coli positive from other E. coli negative among 50 clinical urinary tract infection samples with 96% sensitivity and 100% specificity. Furthermore, we also apply this quantitative detection approach to achieve rapid antimicrobial susceptibility testing within 3 h. This work exhibits easy-to-use protocols, high sensitivity, and short turnaround time for point-of-care testing uses.
Project description:Effective treatment of sexually transmitted infections (STIs) is limited by diagnostics that cannot deliver results rapidly while the patient is still in the clinic. The gold standard methods for identification of STIs are nucleic acid amplification tests (NAATs), which are too expensive for widespread use and have lengthy turnaround times. To address the need for fast and affordable diagnostics, we have developed a portable, rapid, on-cartridge magnetofluidic purification and testing (PROMPT) polymerase chain reaction (PCR) test. We show that it can detect <i>Neisseria gonorrhoeae,</i> the pathogen causing gonorrhea, with simultaneous genotyping of the pathogen for resistance to the antimicrobial drug ciprofloxacin in <15 min. The duplex test was integrated into a low-cost thermoplastic cartridge with automated processing of penile swab samples from patients using magnetic beads. A compact instrument conducted DNA extraction, PCR, and analysis of results while relaying data to the user via a smartphone app. This platform was tested on penile swab samples from sexual health clinics in Baltimore, MD, USA (<i>n</i> = 66) and Kampala, Uganda (<i>n</i> = 151) with an overall sensitivity and specificity of 97.7% (95% CI, 94.7 to 100%) and 97.6% (95% CI, 94.1 to 100%), respectively, for <i>N. gonorrhoeae</i> detection and 100% concordance with culture results for ciprofloxacin resistance. This study paves the way for delivering accessible PCR diagnostics for rapidly detecting STIs at the point of care, helping to guide treatment decisions and combat the rise of antimicrobial resistant pathogens.