Etiologic diagnosis of lower respiratory tract bacterial infections using sputum samples and quantitative loop-mediated isothermal amplification

Abstract

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.

Conflict of interest statement

Competing Interests: In the manuscript, three authors are staffs of Beijing Capitalbio Corporation, which is also named as “National Engineering Research Center for Beijing Biochip Technology", fully owned by the Chinese government. As a basic research, this study is completely funded by the Chinese government. National Engineering Research Center for Beijing Biochip Technology (Capitalbio Corporation) was one of the 23 cooperating institutes for this project. Based on this study, Capitalbio Corporation may develop products in the future. But currently there is no relevant patent or any product under development and in the market. This does not alter the authors’adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. A flow chart of patient recruitment and sample processing.

After admission, we collected 4 sputum samples from each patient. The same samples are used for the parallel qLAMP and culture tests. The results from qLAMP and one-time culture are from the same sample, and those of qLAMP and three-time culture are from the same patient, but not the same sample.

Figure 2. qLAMP and culture result from LRTI patients.

(A) The positive rates (the right vertical axis) of one-time culture (brown bar), three-time culture (blue bar), and quantitative LAMP (yellow bar) for the eight species in the panel (from the left: Ab, A. baumannii; Ec, E. coli; Hi, H. influenzae; Kp, K. pneumoniae; Pa, P. aeruginosa; Sa, S. aureus; Sm, S. maltophilia; and Sp, S. pneumoniae) detected from the number of patients (the left vertical axis). (B) The number of patients (the left vertical axis) who were tested positive for at least one bacterium in one-time culture, three-time culture, and qLAMP. Each bar is the sum of patient with single (blue bar) and multiple (yellow bar) species detected.

Figure 3. Examples of S. pneumonia showing the relationship between qLAMP and culture results (logistic regression) and cutoff determination based on competitive relationship (piece-wise linear regression).

The horizontal axis displays the bacterial natural logarithmic titer in sputum sample. (A) Logistic regression curve (green line). Solid circles indicate patients; they are placed at the top of the chart when being test as positive and at the bottom of the chart when being tested as negative in the culture assays. The height and width of the bars display the frequency and the number of patients being tested positive in cultures, respectively. (B) Piecewise linear regression (black lines) of S. pneumonia in COPD patients. Open circles indicate patients; they are placed at the top of the chart when being PC (Pathogen Candidate) and at the bottom of the chart when NOT being PC.

Figure 4. The percentage of bacteria identified as definite (dark blue) and possible (light blue) causative agents in LRTI patients (for the full bacterial names see the legend of

Figure 2). The patients are classified as: children, ≤14 yr; middle-aged adult, >14 yr but <70 yr; aged, ≥70 yr; AB, acute bronchitis; CAP, community acquired pneumonia; AECOPD, acute exacerbation of COPD; and AEBX, acute exacerbation of bronchiectasis.

Figure 5. The qLAMP-based diagnostic rates in LRTI patients and different subgroups.