Can Wang

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship. Trial Registration ClinicalTrials.gov NCT00567827

Detect early stage lung cancer by a LAMP microfluidic chip system with a real-time fluorescent filter processor

This work presents a promising clinical molecular diagnostics for early stage lung cancer. This novel diagnostic method utilized the loop-mediated isothermal amplification (LAMP), microfluidic chips and a confocal optical detector with a non-linear fluorescent filter processor. An isothermal amplification based microfluidic chip for the early diagnostics of lung cancer was developed and a confocal optical detector was improved by a novel real-time fluorescent filter to sensitively monitor the DNA amplification procedure with high signal to noise ratio and fluorescence collecting ability. Experiment showed that a rapid diagnostic of lung cancer by detecting the existence of the CEA mRNA could be performed in only 5 μL of reaction assay in less than 45 min. While the traditional in-tube RT-PCR set consumed more than 25 μL of the assay and took more than 90 min.

Sensitive sequence-specific molecular identification system comprising an aluminum micro-nanofluidic chip and associated real-time confocal detector

We developed a micro-nanofluidic bioreactor-detector system for isothermal DNA amplification and sensitive real-time detection of the amplified products for sequence-specific molecular identification. Aluminum (Al) chips with a range of volumes from 7.07 μL to 39 nL and an associated real-time confocal optical detector are described. The detector provided highly sensitive fluorescence detection and low background noise. One of the important aspects of the system was the development of a surface processing technique that afforded chips with an inert surface to improve amplification stability in micro-nanoliter reaction assays. The micro-nanofluidic system exhibited more sensitive exponential DNA amplification characteristics than a standard PCR tube amplification system with a volume of 25 μL, the response time was clearly reduced at the same DNA template concentration, and the sensitivity in the number of copies of the DNA template was improved by >600-fold. Efficient amplification of nucleic acid was achieved with as few as three copies of the DNA template. This system may be useful for the development of novel lab-on-a-chip devices and shows promise for single-molecule amplification in droplet assays, with potential applications in nanobiotechnology, nanomedicine, and clinical molecular diagnostics.

A Pilot Study of Quantitative Loop-mediated Isothermal Amplification-guided Target Therapies for Hospital-acquired Pneumonia

Background:It is important to achieve the definitive pathogen identification in hospital-acquired pneumonia (HAP), but the traditional culture results always delay the target antibiotic therapy. We assessed the method called quantitative loop-mediated isothermal amplification (qLAMP) as a new implement for steering of the antibiotic decision-making in HAP. Methods:Totally, 76 respiratory tract aspiration samples were prospectively collected from 60 HAP patients. DNA was isolated from these samples. Specific DNA fragments for identifying 11 pneumonia-related bacteria were amplified by qLAMP assay. Culture results of these patients were compared with the qLAMP results. Clinical data and treatment strategies were analyzed to evaluate the effects of qLAMP results on clinical data. McNemar test and Fishers exact test were used for statistical analysis. Results:The detection of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Stenotrophomonas maltophilia, Streptococcus pneumonia, and Acinetobacter baumannii by qLAMP was consistent with sputum culture (P > 0.05). The qLAMP results of 4 samples for Haemophilus influenzae, Legionella pneumophila, or Mycoplasma pneumonia (MP) were inconsistent with culture results; however, clinical data revealed that the qLAMP results were all reliable except 1 MP positive sample due to the lack of specific species identified in the final diagnosis. The improvement of clinical condition was more significant (P < 0.001) in patients with pathogen target-driven therapy based on qLAMP results than those with empirical therapy. Conclusion:qLAMP is a more promising method for detection of pathogens in an early, rapid, sensitive, and specific manner than culture.