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

Kang Y, Deng R, Wang C, Deng T, Peng P, Cheng X, Wang G, Qian M, Gao H, Han B, Chen Y, Hu Y, Geng R, Hu C, Zhang W, Yang J, Wan H, Yu Q, Wei L, Li J, Tian G, Wang Q, Hu K, Wang S, Wang R, Du J, He B, Ma J, Zhong X, Mu L, Cai S, Zhu X, Xing W, Yu J, Deng M, Gao Z - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Medicine, Peking University People's Hospital, Beijing, People's Republic of China.

ABSTRACT

Unlabelled: 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.

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Related in: MedlinePlus

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.
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pone-0038743-g002: 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.

Mentions: We obtained raw data from 1533 inpatients out of 2986 eligible candidates qualified for the final analysis, based on samples collected from December 2007 to June 2009 (Figure 1). We summarized patient information including hospitals, demographic characteristics, and LRTI diagnoses in Tables S4 and S5. In culture assay, although we found 43 bacterial species and 17 fungi species in total, only the eight species in the pathogen panel are taken into account. For culture assays done in the central laboratories (one-time culture), we have 100 cases (6.52%) tested positive for at least one of the panel members, and for the culture done in local hospitals (three-time culture), we have 266 cases (17.35%) tested positive for at least one of the panel members in at least one of the three-time cultures (Figure 2). For each species, the average confirmation rates among all cultures (four times altogether: one done in the central laboratory and three done in local hospitals) range from 0.17 to 0.50 (Table 1). In other words, it is impossible to detect the same species of bacteria in a sputum sample in all repeated culture assays. The qLAMP and culture results from the central laboratories were never communicated to local hospitals, and the strategies of anti-infectious treatments are only based on the culture results obtained independently. No adverse effects are found either being caused by the protocols or as results of performing qLAMP and routine culture assays on patients during the study.


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

Kang Y, Deng R, Wang C, Deng T, Peng P, Cheng X, Wang G, Qian M, Gao H, Han B, Chen Y, Hu Y, Geng R, Hu C, Zhang W, Yang J, Wan H, Yu Q, Wei L, Li J, Tian G, Wang Q, Hu K, Wang S, Wang R, Du J, He B, Ma J, Zhong X, Mu L, Cai S, Zhu X, Xing W, Yu J, Deng M, Gao Z - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375278&req=5

pone-0038743-g002: 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.
Mentions: We obtained raw data from 1533 inpatients out of 2986 eligible candidates qualified for the final analysis, based on samples collected from December 2007 to June 2009 (Figure 1). We summarized patient information including hospitals, demographic characteristics, and LRTI diagnoses in Tables S4 and S5. In culture assay, although we found 43 bacterial species and 17 fungi species in total, only the eight species in the pathogen panel are taken into account. For culture assays done in the central laboratories (one-time culture), we have 100 cases (6.52%) tested positive for at least one of the panel members, and for the culture done in local hospitals (three-time culture), we have 266 cases (17.35%) tested positive for at least one of the panel members in at least one of the three-time cultures (Figure 2). For each species, the average confirmation rates among all cultures (four times altogether: one done in the central laboratory and three done in local hospitals) range from 0.17 to 0.50 (Table 1). In other words, it is impossible to detect the same species of bacteria in a sputum sample in all repeated culture assays. The qLAMP and culture results from the central laboratories were never communicated to local hospitals, and the strategies of anti-infectious treatments are only based on the culture results obtained independently. No adverse effects are found either being caused by the protocols or as results of performing qLAMP and routine culture assays on patients during the study.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Medicine, Peking University People's Hospital, Beijing, People's Republic of China.

ABSTRACT

Unlabelled: 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.

Show MeSH
Related in: MedlinePlus