Development of rapid, automated diagnostics for infectious disease: advances and challenges

The last 2 years has seen an exponential rise in the amount of research funding made available for the development of rapid diagnostic devices for infectious agents of medical importance. This review reports on several such projects. These highlight the development of fully automated devices for rapid diagnostics, ranging from fully automated real-time PCR-based detection methods to fully automated PCR- and array-based machines for the detection and typing of influenza. This review will also highlight the importance of refocusing work on classical immunoassay techniques, showing how biosensor-based immunoassays can greatly enhance existing assays and at a much reduced cost to molecular-based methods

Microfluidics: innovative approaches for rapid diagnosis of antibiotic-resistant bacteria

Correspondence: Surang Chankhamhaengdecha ([email protected]) The emergence of antibiotic-resistant bacteria has become a major global health concern. Rapid and accurate diagnostic strategies to determine the antibiotic susceptibility profile prior to antibiotic prescription and treatment are critical to control drug resistance. The standard diagnostic procedures for the detection of antibiotic-resistant bacteria, which rely mostly on phenotypic characterization, are time consuming, insensitive and often require skilled personnel, making them unsuitable for point-of-care (POC) diagnosis. Various molecular techniques have therefore been implemented to help speed up the process and increase sensitivity. Over the past decade, microfluidic technology has gained great momentum in medical diagnosis as a series of fluid handling steps in a laboratory can be simplified and miniaturized on to a small platform, allowing marked reduction of sample amount, high portability and tremendous possibility for integration with other detection technologies. These advantages render the microfluidic system a great candidate to be developed into an easy-to-use sample-to-answer POC diagnosis suitable for application in remote clinical settings. This review provides an overview of the current development of microfluidic technologies for the nucleic acid based and phenotypic-based detections of antibiotic resistance

Development and integration of simplified real-world to chip interfaces for use in the detection of infectious diseases

Bacterial-based infectious disease, such as sexually transmitted infections and hospital-acquired infections, present a worldwide burden on healthcare issues. To control the spread of infection and to inform clinical treatment, rapid point of care (POC) diagnosis is required. Although some are currently available, these are commonly limited by a requirement for sample processing prior to analysis and a requirement for user intervention. Novel real world-to-chip interfaces are required which can receive a sample with little or no pre-processing and should be manufactured with minimal cost. In addition, manufacturing protocols should ideally be developed to allow easy adjustment of design for customising to process a wide variety of sample types and volumes.Here, focussing on bacterial-based POC diagnostics, a microfluidic platform has been developed which holds the potential to receive a variety of sample types for the detection of infectious organisms by implementing multiple sample-to-chip interfaces. The platform consists of a glass microfluidic device which is incorporated in to a custom-made integrated genetic analyser (IGA) for sample processing.A series of interfacing substrates were investigated using two types of porous silica and the biopolymer chitosan (α(1→4)-linked 2-amino-2-deoxy-β-D-glucopyranose) as contributing materials. For analysis of urine samples, a porous silica monolith, synthesised from tetramethyl orthosilicate, was developed, capable of receiving and processing human urine samples (≈ 150 μl) for DNA capture and purification. Due to the nature of synthesis, these monoliths hold the potential for resizing and shaping, dependent on the sample volume and for integration to downstream steps, such as polymerase chain reaction (PCR) amplification. The monolith was first optimised structurally using flow systems made from monoliths encased in heat shrink wrap and was incorporated in to a microfluidic device by way of disk. The latter was achieved by sealing the monolith in place with a sondary porous silica phase synthesised from potassium silicate, creating a dual porous silica (DPS) real world interface. The DNA extraction efficiencies of monolithic flow systems and the DPS system were 51 % and 44 % respectively. The DPS was shown to provide DNA of sufficient quality and integrity to support PCR amplification for both Chlamydia trachomatis and Neisseria gonorrhoea target sequences. The system did however lack sensitivity (1.3 x 10¯³ ng DNA μl¯¹ urine), when compared with systems of similar applications in the literature, likely due to large elution volumes (> 20 μl) and/or ethanol carryover. In addition, chitosan was introduced to the silica surface of the monolith as an alternative methodology for DNA extraction by anion exchange. The system provided DNA extraction efficiencies of 40 % and DNA was subsequently amplified by PCR.Using an alternative application, an investigation was also carried out in to the analysis of small volume blood samples (≤ 6 μl) for use in the same system. This was achieved by implementing a Phusion® blood direct kit in to a glass microfluidic device to amplify gDNA of bacterial target, methicillin resistant Staphylococcus aureus (MRSA). The system was shown to amplify DNA in 6 μl blood (0.083 ng μl¯¹) off-chip. It was then demonstrated to work on-chip in a 12.5 μl glass chamber, using a Peltier system for thermal cycling.For use in conjunction with all interfaces, an IGA with a built in DNA separation/ detection system, based on plug injection, capillary electrophoresis separation and fluorescence detection has been shown to reproducibly report the presence of target DNA sequences, against that of a custom-made size ladder. The detection of a 107 bp generic Staphylococcus aureus marker and 532 bp sequence from the mecA gene unique to MRSA were detected in 20 and 25 min, respectively. Importantly, the detection system is designed to integrate directly to upstream steps and has a Peltier element fitted for thermal cycling.The work described here contributes towards a platform which offers the opportunity to tackle a number of diagnostic applications in one fit-for-all instrument

Fully integrated microsystem for bacterial genotyping

Methods for bacterial detection and identification has garnered renewed interest in recent years due to the infections they may cause and the antimicrobial resistances they can develop, the potential for bioterrorism threats and possible contamination of food/water supplies. Therefore, the rapid, specific and accurate detection of pathogens is crucial for the prevention of pathogen-related disease outbreaks and facilitating disease management as well as the containment of suspected contaminated food and/or water supplies. In this dissertation an integrated modular-based microfluidic system composed of a fluidic cartridge and a control instrument has been developed for bacterial pathogen detection. The integrated system can directly carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and can detect sequence variations in selected genes to allow for the identification of the bacterial species and even its strain. The unique aspect of this fluidic cartridge is its modular format with a task-specific module interconnected to a fluidic motherboard to permit the selection of a material appropriate for the given processing step(s). In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the fluidic cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and assembled into a small footprint instrument. The fluidic cartridge was capable of performing cell lysis, solidphase extraction of genomic DNA from the whole cell lysate, continuous flow PCR amplification of specific gene fragments, continuous flow ligase detection reaction to discriminate sequence variations and universal DNA array readout, which consisted of DNA probes patterned onto a planar polymer waveguide for evanescent excitation. The performance of the fluidic system was demonstrated through its successful application to the genetic detection of bacterial pathogens, such as Escherichia coli O157:H7, Salmonella, methicillin-resistant Staphylococcus aureus and multi-drug resistant Mycobacterium tuberculosis, which are major threats for global heath. The modular system, which could successfully identify several strains of bacteria in \u3c40 min with minimal human intervention and also perform strain identification, represents a significant contribution to pathogen detection

Multiplexed microfluidic loop-mediated isothermal amplification of the 16s rRNA gene for the diagnosis of neonatal sepsis in resource-limited environments

Bacterial infection, or sepsis, places a disproportionately high burden on newborns in developing countries. This is due in part to a lack of diagnostic tools suitable for sustainable use in resource-limited nurseries. One potential vehicle for a new diagnostic assay is loop-mediated isothermal amplification (LAMP), a high-yield DNA amplification method. LAMP has been used to detect single genes from bacteria in blood serum samples to aid in sepsis diagnosis. While specific, this approach can only provide detection for one species at a time. LAMP could be adapted to detect a broader set of bacteria, while retaining a degree of specificity that allows clinicians to begin directed antimicrobial therapy. Described herein is the successful design of a novel group of oligonucleotide LAMP primer sets that specifically bind to regions of the 16s rRNA gene of four bacterial Orders. These regions lie on the transition area between sequences that are highly conserved and those that are hypervariable. This allows each primer set to be specific for one Order of bacteria. When primers bind, amplification occurs, and the large quantities of DNA produced could be detected using a fluorescent indicator. The four separate LAMP reactions could be multiplexed on a microfluidic chip to provide clinicians with a two-step sepsis-diagnosis technique that could give a result in only one hour. Future studies should examine fluorescent indicators, reaction multiplexing, and specificity of primers to recognize more species

Utility and Impact of Detecting Clinically Important Bacteria with Small-Scale and Automated Nucleic-Acid Amplification Assays

During the last decade, nucleic acid-based amplification techniques (NAATs) have revolutionized the way clinical microbiology laboratories diagnose human pathogens. Modern automated NAATs require considerably less hands-on time and testing is much simpler than with conventional detection methods. The combination of ease of performance and speed, has made real-time NAATs appealing alternatives to conventional culture-based or immunoassay-based testing methods for diagnosing various infectious diseases. However, in this era of implementing new technologies, it is crucial to focus not only upon the possibilities but also upon the pitfalls of the technology. Failure to do so may increase the cost of implementation, and put the new technology at risk of losing reputation in the eyes of the clinicians. This thesis deals with the basic principles behind modern NAATs and the implementation and utility of some of these modern assays in clinical diagnostics. The thesis consists of six studies on three new fully automated NAAT platforms, the BD Max, the GenomEra CDX, and the GenRead system. All platforms were used for the screening of toxigenic Clostridium difficile in faecal specimens in comparison with the routine laboratory methods. In addition, the GenomEra CDX system was used for the detection of Staphylococcus aureus and the marker of methicillin resistance from various sample matrixes, and the detection of Streptococcus pneumoniae from blood cultures. All assays showed excellent sensitivity and specificity for the target microbes. Moreover, all platforms decreased the analysis time significantly as compared to conventional laboratory methods, although variation between the NAAT test systems were seen. As its best, the total turnaround-time was less than 30 minutes with the GenRead, 55 minutes with the GenomEra, and 90 minutes with the BD Max system. The platforms had different sample throughput capacity and space requirement, as well. The lightweight and battery-powered GenRead instrument with isothermal NAAT based assay proved to be most flexible system for clinical diagnostics, enabling mobile analytics in both laboratory and near patient. Molecular techniques such as real-time PCR and isothermal amplification combined with modern robotics can provide significant advantage in laboratory diagnostics for detection of pathogenic bacteria. In this study, the three NAAT platforms proved to be suitable for rapid testing of C. difficile, methicillin-susceptible and -resistant S. aureus, and S. pneumoniae from various sample types.Viime vuosikymmenen aikana nukleiinihappopohjaiset monistustekniikat ovat mullistaneet kliinisesti merkittävien patogeenien mikrobiologista laboratoriodiagnostiikkaa. Modernit automatisoidut nukleiinihaponosoituslaitteistot vaativat huomattavasti vähemmän käsityöaikaa ja testaus on yksinkertaisempaa kuin tavanomaisilla laboratoriomenetelmillä. Helppokäyttöisyys ja nopeus on tehnyt reaaliaikaisista nukleiinihappotekniikoista houkuttelevia vaihtoehtoja perinteisille viljely- tai antigeenipohjaisille analyysimenetelmille erilaisten tartuntatautien diagnostiikkaan. Uusien tekniikoiden ilmaantuessa on kuitenkin tärkeää keskittyä paitsi niiden tuomiin mahdollisuuksiin myös niissä piileviin haasteisiin. Mikäli näin ei tehdä, saattaa huolimaton käyttöönotto lisätä laboratorion kustannuksia ja asettaa uuden tekniikan vaaraan menettää arvoaan asiantuntijoiden silmissä. Tämä väitöskirjatyö käsittelee modernien nukleiinihaponosoitusmenetelmien perusperiaatteita sekä joidenkin näiden tekniikoiden hyödyntämistä kliinisen mikrobiologian diagnostiikassa. Väitöskirjatyö koostuu kuudesta osatutkimuksesta, jotka käsittelevät kolmea automatisoitua nukleiinihaponosoituslaitteistoa; BD Max:ia, GenomEra CDX:ää ja GenRead:iä. Kaikkia kolmea laitetta käytettiin toksigeenisen Clostridium difficilen seulonnassa ulostenäytteistä ja saatuja tuloksia verrattiin kliinisten laboratorioiden käytössä oleviin rutiinimenetelmiin. Tämän lisäksi GenomEra CDX -laitteistoa hyödynnettiin Staphylococcus aureuksen ja metisilliiniresistenssigeenin havaitsemiseen erilaisista näytemateriaaleista sekä Streptococcus pneumoniaen havaitsemiseen veriviljelynäytteistä. Kaikki testisysteemit osoittivat erinomaista herkkyyttä ja tarkkuutta kohdemikrobien suhteen. Lisäksi testatut laitteistot pienensivät analyysiaikaa merkittävästi tavanomaisiin laboratoriomenetelmiin verrattuna, joskin tässä testijärjestelmien välillä haivatiin selvää vaihtelua. Parhaimmillaan kokonaistestiaika oli alle 30 minuuttia GenRead-laitteella, 55 minuuttia GenomEra-laitteella ja 90 minuuttia BD Max -laitteella. Myös laitteistojen näyteanalyysikapasiteetissä ja tilavaatimuksessa havaittiin eroavuuksia. Kevyt ja akkukäyttöinen GenRead-laite, jossa hyödynnettiin isotermistä nukleiinihaponosoitusmääritystä, osoittautui joustavimmaksi kliiniseen diagnostiikkaan mahdollistaen mukana kuljetettavan analyysiyksikön, joka soveltuu sekä laboratorioon että lähellä potilasta tehtävään analytiikkaan. Molekyylitekniikat, kuten reaaliaikainen PCR ja isoterminen nukleiinihaponosoitus yhdistettynä nykyaikaiseen robotiikkaan, voivat mahdollistaa merkittävän diagnostisen hyödyn patogeenisten bakteerien analytiikkaan. Tässä väitöskirjatyössä tutkitut kolme nukleiinihappopohjaista testisysteemiä osoittautuivat erittäin soveltuviksi C. difficilen, metisilliiniherkkien ja -resistenttien S. aureus sekä S. pneumoniae -bakteerien nopeaan tunnistukseen useista eri näytetyypeistä

Microfluidic pathogen detection, based on continuous bead beating. DNA concentration and aliquoting for multiplex qPCR detection

108 p.El objetivo de este trabajo es la detección de agentes patógenos por medio de su ADN en un sistema microfluídico que funciona de forma automática. El sistema consta de tres pasos: en el primero se lisan los microorganismos para extraer su ADN. En el segundo, el ADN extraído se concentra gracias a partículas magnéticas. De esta forma, por un lado se consigue mejorar la sensibilidad de la detección y por otro se deshacen los restos celulares originados en el paso previo. Por último, el extracto se divide en dos alícuotas y el ADN se detecta en paralelo utilizando la técnica de qPCR. Mediante la detección en paralelo, se pueden hacer medidas en duplicado o bien detectar dos patógenos a la vez

Genotyping Approaches for Identification and Characterization of Staphylococcus aureus

Genotyping methods are vital epidemiological tools for discriminating different bacterial isolates within same species, which in turn provide useful data in tracing source of infection and disease management. There have been a revolutionary efforts in ways to distinguish between bacterial types and subtypes at molecular level utilizing DNA in the genomes. Notably, the growth of various DNA typing methods has provided innovative apparatuses for improved surveillance and outbreak investigation. Thus, early identification and genotyping are indispensable as resources for managing therapeutic treatment and the control of rapid expansion of clinically important bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) has been in a great attention due to its contagious nature and subjected to various typing analyses. Thus, in this chapter, we aimed to review the contribution of various genotyping methods of commonly used as well as those unique to staphylococci in understanding its epidemiology, infection and dissemination pattern, and to provide an impression of specific advantages and disadvantages of each tool

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Loop Mediated Isothermal Amplification (LAMP)

Staphylococcus aureus is one of the most common pathogens that cause a wide range of infections ranging from skin and soft tissue infections to invasive, life threatening infections. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) substantially increased healthcare burdens associated with Staphylococcal infections because of high morbidity and mortality and increasing the need for efficient and cost-effective screening methods, for high-risk patients. The objective of this study is to develop two molecular methods, real-time PCR and loop-mediated isothermal amplification (LAMP), and validate them following Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) standards. The real time PCR assay was developed targeting mecA, mecC, nuc, and coa to detect S. aureus and methicillin-resistance. The assay had high precision, a linear range of 104-108 CFU/ml, and 95% accuracy. The assay detects MRSA, MSSA, MR-CoNS, and MS CoNS. The LAMP assay was developed targeting the same genes; however, its lower limit of detection was 106 CFU/ml, which was much higher than that of the real-time PCR assay. Additional studies are required to optimize the performance characteristics of the LAMP assay further. Nevertheless, the real-time PCR assay developed in this study will be useful for the detection of MRSA in a cost-effective manner