Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection

Abstract

Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70–120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.

Clinical evaluation of the loop-mediated isothermal amplification assay for the detection of common lower respiratory pathogens in patients with respiratory symptoms

“…Several LAMP-based protocols have been successfully developed and applied for the effective detection of pathogenic organism. [22–24] In our study, the LAMP assay was performed for the detection of pathogenic bacteria in patients with LRTI, the bacteria amount excreted from lower respiratory tract was very little or absent, the detectable rate of both the sputum or bronchoalveolar lavage fluid samples was relatively too low to diagnose the LRTI by culture method. On the other side, the diagnostic method used in this study, which combined isothermal amplification methods and microfluidics chip, displaying high specificity and sensitivity.…”

“…[17–19] The LAMP reaction employs a bacillus stearothermophilus DNA polymerase (Bst) with strand-displacement activity and a set of suitable primers that bind to 6 distinct regions on the target DNA, which makes the initial denaturation step obsolete and can be performed under isothermal conditions without costly equipment such as thermal cycler equipment. [20–22] Based on these properties, the LAMP approaches have been widely used for the detection of pathogens in clinical diagnostics as a rapid, accurate and cost-effective method. [23–26] Additionally, it was demonstrated that LAMP assay can be combined with microfluidic chips for improved practicability in clinical diagnostics and it can be appropriate in primary hospitals.…”

Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of human and plant diseases

“…Isothermal amplification methods such as LAMP have been widely used in the detection of pathogens in sputum samples ( Bentaleb et al, 2016 ; Etchebarne et al, 2017 ; Wang et al, 2016 ). For tuberculosis detection, Bentaleb et al (2016) demonstrated that the LAMP assay successfully amplified target pathogen from homogenized sputum samples with 4% NaOH solution.…”

“…In this device, the bacterial cells were trapped on the paper, and then the captured cells were directly used for in-situ HDA amplification in the tip. Similarly, Etchebarne et al (2017) demonstrated a direct LAMP assay without DNA extraction from concentrated bacterial cells in urine samples. However, low detection sensitivity was observed for the direct amplification assays without nucleic acid extraction ( Wand et al, 2018 ).…”

Water and Microbial Monitoring Technologies towards the Near Future Space Exploration

“…Positive reactions can be visualised by naked eye (i.e., without post-amplification steps) following the increase of sample turbidity or colour owing to the addition of fluorescent dyes (Notomi et al, 2015). Being less sensible than PCR to inhibition and not significantly influenced by non-target DNA, direct LAMP assays are currently used for point of use diagnostic in clinical and environmental settings, with good analytical performances on moderately pre-treated samples and in a range of temperature, pH and elongation times (Etchebarne et al, 2017;Samhan et al, 2017). Low amount of DNA can be amplified up to generating 10 9 copies within 1 h and producing as final amplification product a complex stem-loop DNA, with several inverted repeats of the target and cauliflower-like structures.…”