Diseases caused by pathogenic microorganisms including bacteria and viruses can cause serious medical issues including death and result in huge economic losses. Despite the myriad of recent advances ...in the rapid and accurate detection of pathogens, large volume clinical samples with a low concentration of pathogens continue to present challenges for diagnosis and surveillance. We here report a simple and label-free approach via homobifunctional imidoesters (HIs) with a microfluidic platform (SLIM) to efficiently enrich and extract pathogens at low concentrations from clinical samples. The SLIM system consists of an assembled double microfluidic chip for streamlining large volume processing and HIs for capturing pathogens and isolating nucleic acids by both electrostatic and covalent interaction without a chaotropic detergent or bulky instruments. The SLIM system significantly increases the enrichment and extraction rate of pathogens (up to 80% at 10 CFU (colony forming unit) in a 1 mL volume within 50 min). We demonstrated its clinical utility in large sample volumes from 46 clinical specimens including environmental swabs, saliva, and blood plasma. The SLIM system showed higher sensitivity with these samples and could detect pathogens that were below the threshold of detection with other methods. Finally, by combining our SLIM approach with an isothermal optical sensor, pathogens could be detected at a very high sensitivity in blood plasma samples within 80 min via enrichment, extraction and detection steps. Our SLIM system thus provides a simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform for use with large volume clinical samples and would thus have significant utility for various infectious diseases.
•SLIM system significantly increases the enrichment and extraction rate of pathogens.•Demonstrated its clinical utility in large sample volumes from 46 clinical specimens.•A simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform.
Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA ...in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.
Rapid identification of emerging infectious pathogens is crucial for preventing public health threats. Various pathogen detection techniques have been introduced; however, most techniques are ...time-consuming and lack multiple-target detection specificity. Although multiple-target detection techniques can distinguish emerging infectious pathogens from related pathogens, direct amplification methods have not been widely examined. Here, we present a novel arch-shaped multiple-target sensor capable of rapid pathogen identification using direct amplification in clinical samples. In this study, an arch-shaped amplification containing primer sequences was designed to rapidly amplify multiple targets. Further, the sensing platform allowed for sensitive and specific detection of human coronavirus, Middle East respiratory syndrome, Zika virus, and Ebola virus down to several copies. This platform also simultaneously distinguished between Middle East respiratory syndrome and human coronavirus in clinical specimens within 20 min. This arch-shaped multiple-target sensing assay can provide rapid, sensitive, and accurate diagnoses of emerging infectious diseases in clinical applications.
•A novel arch-shaped multiple-target sensing capable of rapid pathogen identification.•An arch-shaped amplification containing primer sequences to rapidly amplify multiple targets.•Allowed sensitive and specific detection of MERS, ZIKV, and EBOV in 20 min.•Validated clinical utility of the platform in MERS patient samples.
•We developed a nanobiosensing platform combining with microfluidic enrichment for rapid and sensitive detection of pathogens.•Salmonella Typhimurium was detected in urine samples in real-time using ...a label-free method.•Sensitivity was improved by microfluidic enrichment.•The whole experiment was completed within 100 min.
Urinary tract infections are among the most common bacterial infections in humans, causing relapses and acute prostatitis as well as significant morbidity and high medical costs. In bacteria with low abundance in urological samples, which contain various contaminating factors, sample analyses should be conducted with meticulous care; therefore, emerging technologies can facilitate the characterization of disease-causing bacteria with more sensitivity, rapidity, and ease of use. In this study, we developed a highly sensitive nanobiosensor based on isothermal amplification combining microfluidic enrichment using a concanavalin A–functionalized microchannel with asymmetric herringbone groove arrays for rapid detection of pathogens. After optimization of enrichment and detection conditions, we demonstrated that Salmonella enterica serotype Typhimurium could be detected in urine samples (10 mL) at a concentration as low as 5 CFU/mL in real-time using a label-free method. Moreover, the use of microfluidic enrichment improved the sensitivity by 1.76 orders of magnitude. The whole experiment was completed within 100 min. This developed method has the potential to provide a simple, rapid, sensitive diagnostic platform and can be used in practical applications for detection of Salmonella or other pathogenic bacteria, causing urinary tract infections.
Enrichment cultivation was performed after a seashore soil polluted by crude oil was inoculated into a basal medium with low-molecular-weight polyethylene (LMWPE) powder as the sole carbon source. ...From the whole DNAs, alkane monooxygenase gene (alkB), which has been reported to participate in the degradation of polyethylene, was measured with quantitative real-time PCR to monitor the abundance of polyethylene degrading bacteria in the culture. The change in the ratio of alkB/16S rRNA in the culture broth as a function of enrichment cultivation time was measured and used as an index for the ratio of microbes with alkB to those in the entire bacterial community in the culture broth. Through 16S rRNA sequence analysis, the bacterial community was analyzed at the genus level. With this technique, the changes in the community of soil microbes and their diversity as a function of enrichment cultivation time were examined. In addition, improvement in the LMWPE degradation ability of the bacterial community due to LMWPE enrichment cultivation was analyzed through biodegradability testing under controlled compost conditions.
•The amount of 16S rRNA decreased while the alkB/16S rRNA ratio increased with increase of LMWPE enrichment cultivation.•Abundance of genera Curtobacterium, Gordonia and Rhodococcus increased over the course of enrichment cultivation.•The LMWPE biodegradability was higher in the sterilized compost inoculated with the LMWPE enrichment culture experienced soil.
Versatile, simple and efficient sample preparation is desirable for point-of-care testing of emerging diseases such as zoonoses, but current sample preparation assays are insensitive, ...labour-intensive and time-consuming and require multiple instruments. We developed a single-tube sample preparation approach involving direct pathogen enrichment and extraction from human specimens using diatomaceous earth (DE). Amine-modified DE was used to directly enrich a zoonotic pathogen, Brucella, in a large sample volume. Next, a complex of amine-modified DE and dimethyl suberimidate was used for nucleic acid extraction from the enriched pathogen. Using our single-tube approach, the pathogen can be enriched and extracted within 60min at a level of 1 colony formation unit (CFU) from a 1ml sample volume in the same tube. The performance of this approach is 10–100 times better than that of a commercial kit (102 to 103CFU/ml) but does not require a large centrifuge. Finally, we combined the single-tube approach with a bio-optical sensor for rapid and accurate zoonotic pathogen detection in human urine samples. Using the combination system, Brucella in human urine can be efficiently enriched (~ 8-fold) and the detection limit is enhanced by up to 100 times (1CFU/ml bacteria in urine) compared with the commercial kit. This combined system is fast and highly sensitive and thus represents a promising approach for disease diagnosis in the clinical setting.
•A single-tube sample preparation approach involving direct pathogen enrichment and extraction.•Pathogen can be enriched and extracted within 60min at a level of 1 colony.•Combination of the single-tube approach with a bio-optical sensor for rapid and accurate zoonotic pathogen detection.•This system can be efficiently enriched (~ 8-fold) and the detection limit is enhanced by up to 100 times.
Cell‐free nucleic acids (cfNAs) are emerging diagnostic biomarkers for monitoring the treatment and recurrence of cancers. In particular, the biological role and clinical usefulness of cfNAs obtained ...from the plasma of patients with various cancers are popular and still intensely explored, yet most studies are limited by technical problems during cfNA isolation. A dimethyl dithiobispropionimidate (DTBP)‐based microchannel platform that enables spontaneous cfNA capture in 15 min with minimal cellular background and no requirements for use of bulky instruments is reported first. This platform identified KRAS and BRAF hot‐spot mutations following cfDNA isolation from the blood plasma and tissues obtained from 30 colorectal cancer patients. The correlation of mutations between the primary tissues and plasma from the patients was high using this platform with whole genome sequencing compared to the spin‐column method. This platform can also be combined with various detection approaches (biooptical sensor, Sanger sequencing, and polymerase chain reaction (PCR)) for rapid, simple, low‐cost, and sensitive circulating tumor DNA detection in blood plasma. The efficiency and versatility of this platform in isolating cfNAs from liquid biopsies has applications in cancer treatment and precision medicine.
Dimethyl dithiobispropionimidate platform enables spontaneous cell‐free nucleic acid (cfNA) capture in 15 min with less cellular background and no requirement for bulky instruments. This platform can be combined with various detection approaches for rapid, simple, low‐cost, and sensitive circulating tumor DNA detection in blood plasma. The efficiency and versatility of this platform in isolating cfNAs from liquid biopsies can find various cancer applications.
Serologic diagnosis is one of the most widely used diagnostic methods for Q fever, but the window period in antibody response of 2 to 3 weeks after symptom onset results in significant diagnostic ...delay. We investigated the diagnostic utility of Q fever PCR from formalin-fixed liver tissues in Q fever patients with acute hepatitis.
We reviewed the clinical and laboratory data in patients with Q fever hepatitis who underwent liver biopsy during a 17-year period, and whose biopsied tissues were available. We also selected patients who revealed granuloma in liver biopsy and with no Q fever diagnosis within the last 3 years as control. Acute Q fever hepatitis was diagnosed if two or more of the following clinical, serologic, or histopathologic criteria were met: (1) an infectious hepatitis-like clinical feature such as fever (≥ 38°C) with elevated hepatic transaminase levels; (2) exhibition of a phase II immunoglobulin G (IgG) antibodies titer by IFA of ≥ 1:128 in single determination, or a four-fold or greater rise between two separate samples obtained two or more weeks apart; (3) histologic finding of biopsy tissue showing characteristic fibrin ring granuloma.
A total of 11 patients with acute Q fever hepatitis were selected and analyzed. Of the 11 patients, 3 (27%) had exposure to zoonotic risk factors and 7 (63%) met the serologic criteria. Granulomas with either circumferential or radiating fibrin deposition were observed in 10 cases on liver biopsy and in 1 case on bone marrow biopsy. 8 (73%) revealed positive Coxiella burnetii PCR from their formalin-fixed liver tissues. In contrast, none of 10 patients with alternative diagnosis who had hepatic granuloma revealed positive C. burnetii PCR from their formalin-fixed liver tissues.
Q fever PCR from formalin-fixed liver tissues appears to be a useful adjunct for diagnosing Q fever hepatitis.
Background: Sexually transmitted diseases (STDs) are common infectious diseases in humans transmitted through unprotected sexual activities. In South Korea, despite the high annual incidence of STDs, ...detailed examinations of pathogen-specific factors and causes for delays in diagnosis and treatment are still lacking. Furthermore, STD prevalence patterns and important pathogen-specific factors remain unclear. Herein, we retrospectively analyzed the epidemiology of STDs in South Korea in 2019 by analyzing the association of pathogen-specific infection patterns with factors such as sex, age, region, and month. Methods: We obtained the STD test results of 172,973 individuals from the Seoul Clinic Laboratory in 2019, most of whom had multiple infections; hence, 275,296 STD-positive cases were included in this analysis. Through deoxyribonucleic acid (DNA) amplification, they were categorized by pathogen type. Subsequently, they were further classified by month, region, and age while concurrently being stratified according to sex. Results: Among the 12 pathogens detected in this study, Gardnerella vaginalis had the highest prevalence, with 92,490 cases in both sex groups; moreover, many of them were concurrently infected by two or more pathogens. The prevalence of STDs did not differ according to month or region. Conversely, the pathogen-specific prevalence rates significantly differed according to age. Older adults had higher prevalence rates of Chlamydia trachomatis, Trichomonas vaginalis, Candida albicans, and herpes simplex virus type 1 infections than younger adults. Conclusion: These pathogen-specific prevalence patterns provide information that helps to understand population vulnerability according to region and age and helps develop STD prevention and treatment strategies in South Korea.
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