Infectious Disease Diagnostics Research Articles

Smartphone-assisted detection of nucleic acids by light-harvesting FRET-based nanoprobe

Point-of-care assays for optical detection of biomolecular markers attract growing attention, because of their capacity to provide rapid and inexpensive diagnostics of cancer and infectious diseases. Here, we designed a nanoprobe compatible with a smartphone RGB camera for detection of nucleic acids. It is based on light-harvesting polymeric nanoparticles (NPs) encapsulating green fluorescent donor dyes that undergo efficient Förster Resonance Energy Transfer (FRET) to red fluorescent acceptor hybridized at the particle surface. Green-emitting NPs are based on rhodamine 110 and 6G dyes paired with bulky hydrophobic counterions, which prevent dye self-quenching and ensure efficient energy transfer. Their surface is functionalized with a capture DNA sequence for cancer marker survivin, hybridized with a short oligonucleotide bearing FRET acceptor ATTO647N. Obtained 40-nm poly(methyl methacrylate)-based NP probe, encapsulating octadecyl rhodamine 6G dyes with tetrakis(perfluoro-tert-butoxy)aluminate counterions (~6000 dyes per NP), and bearing 65 acceptors, shows efficient FRET with >20% quantum yield and a signal amplification (antenna effect) of 25. It exhibits ratiometric response to the target DNA by FRET acceptor displacement and enables DNA detection in solution by fluorescence spectroscopy (limit of detection 3 pM) and on surfaces at the single-particle level using two-color fluorescence microscopy. Using a smartphone RGB camera, the nanoprobe response can be readily detected at 10 pM target in true color and in red-to-green ratio images. Thus, our FRET-based nanoparticle biosensor enables detection of nucleic acid targets using a smartphone coupled to an appropriate optical setup, opening the way to simple and inexpensive point-of-care assays.

Complete genome sequences of Streptococcus pyogenes type strain reveal 100%-match between PacBio-solo and Illumina-Oxford Nanopore hybrid assemblies

We present the first complete, closed genome sequences of Streptococcus pyogenes strains NCTC 8198T and CCUG 4207T, the type strain of the type species of the genus Streptococcus and an important human pathogen that causes a wide range of infectious diseases. S. pyogenes NCTC 8198T and CCUG 4207T are derived from deposit of the same strain at two different culture collections. NCTC 8198T was sequenced, using a PacBio platform; the genome sequence was assembled de novo, using HGAP. CCUG 4207T was sequenced and a de novo hybrid assembly was generated, using SPAdes, combining Illumina and Oxford Nanopore sequence reads. Both strategies yielded closed genome sequences of 1,914,862 bp, identical in length and sequence identity. Combining short-read Illumina and long-read Oxford Nanopore sequence data circumvented the expected error rate of the nanopore sequencing technology, producing a genome sequence indistinguishable to the one determined with PacBio. Sequence analyses revealed five prophage regions, a CRISPR-Cas system, numerous virulence factors and no relevant antibiotic resistance genes. These two complete genome sequences of the type strain of S. pyogenes will effectively serve as valuable taxonomic and genomic references for infectious disease diagnostics, as well as references for future studies and applications within the genus Streptococcus.

Evaluation of a commercial exogenous internal process control for diagnostic RNA virus metagenomics from different animal clinical samples

Metagenomic next generation sequencing (mNGS) is increasingly recognized as an important complementary tool to targeted human and animal infectious disease diagnostics. It is, however, sensitive to biases and errors that are currently not systematically evaluated by the implementation of quality controls (QC) for the diagnostic use of mNGS. We evaluated a commercial reagent (Mengovirus extraction control kit, CeraamTools, bioMérieux) as an exogenous internal control for mNGS. It validates the integrity of reagents and workflow, the efficient isolation of viral nucleic acids and the absence of inhibitors in individual samples (verified using a specific qRT-PCR). Moreover, it validates the efficient generation of viral sequence data in individual samples (verified by normalized mengoviral read counts in the metagenomic analysis). We show that when using a completely random metagenomics workflow: (1) Mengovirus RNA can be reproducibly detected in different animal sample types (swine feces and sera, wild bird cloacal swabs), except for tissue samples (swine lung); (2) the Mengovirus control kit does not contain other contaminating viruses that may affect metagenomic experiments (using a cutoff of minimum 1 Kraken classified read per million (RPM)); (3) the addition of 2.17 × 106Mengovirus copies/mL of sample does not affect the virome composition of pig fecal samples or wild bird cloacal swab samples; (4) Mengovirus Cq values (using as cutoff the upper limit of the 99 % confidence interval of Cq values for a given sample matrix) allow the identification of samples with poor viral RNA extraction or high inhibitor load; (5) Mengovirus normalized read counts (cutoff RPM > 1) allow the identification of samples where the viral sequences are outcompeted by host or bacterial target sequences in the random metagenomic workflow. The implementation of two QC testing points, a first one after RNA extraction (Mengoviral qRT-PCR) and a second one after metagenomic data analysis provide valuable information for the validation of individual samples and results. Their implementation in addition to external controls validating runs or experiments should be carefully considered for a given sample type and workflow.

Electrochemical Diagnostics for Bacterial Infectious Diseases.

Bacterial infections are urgent threats to human health, especially in light of rising rates of antibiotic resistance, and their ubiquity demands the development of efficient diagnostic platforms. Electrochemical biosensors for point-of-care testing are garnering interest due to their speed, sensitivity, and selectivity as well as their inexpensive, user-friendly operation. These biosensors have the potential to make significant commercial and clinical impacts. In this Viewpoint, we discuss recent advances in the electrochemical detection of pathogenic bacteria using both direct and alternating current. We focus on platforms that detect whole microbes, as these sensors are specific, fast, and easy to operate.

Real-time Colorimetric Quantitative Molecular Detection of Infectious Diseases on Smartphone-based Diagnostic Platform

Rapid diagnostics of infectious diseases and accurate identification of their causative pathogens play a crucial role in disease prevention, monitoring, and treatment. Conventional molecular detection of infectious pathogens requires expensive equipment and well-trained personnel, thus limiting its use in centralized clinical laboratories. To address this challenge, a portable smartphone-based quantitative molecular detection platform, termed “smart connected pathogen tracer” (SCPT), has been developed for pathogen monitoring and disease surveillance. The platform takes advantage of synergistically enhanced colorimetric loop-mediated isothermal amplification (LAMP) assay and smartphone-based color analysis, enabling simple, rapid and reliable nucleic acid quantification without need for expensive fluorescence detection equipment. The SCPT platform has been successfully applied to quantitatively detect: i) HPV DNA in saliva and clinical vaginal swab samples, and ii) HIV RNA in plasma samples with comparable sensitivity to state-of-art machine. It has also been demonstrated for disease spatiotemporal mapping and pathogen tracking by wireless connection and web-based surveillance. Such simple, cost-affordable, portable molecular detection platform has great potential for on-site early disease detection, remote healthcare monitoring, and epidemic surveillance.

Fe3O4@Au SERS tags-based lateral flow assay for simultaneous detection of serum amyloid A and C-reactive protein in unprocessed blood sample

Accurate detection of mutiple biomarkers in unprocessed blood via lateral flow immunoassay (LFA) strips remains challenging. Herein, we reported a novel surface-enhanced Raman scattering (SERS)-based LFA strip for ultrasensitive and simultaneous analysis of dual infection biomarkers by using functionalized Fe3O4@Au magnetic nanoparticles (MNPs). Antibody-modified Fe3O4@Au MNPs were designed as SERS nanotags and efficient enrichment tool. The target infection biomarkers were quickly captured and enriched from the blood sample by Fe3O4@Au nanotags. After washing to remove impurities, Fe3O4@Au nanotags acted as high-performance SERS reporters for quantitative detection of the target in the LFA strip. Through this strategy, the proposed Fe3O4@Au based LFA allowed quantified analysis of target infection biomarkers in unprocessed blood samples with the limits of detection of 0.1 ng/mL and 0.01 ng/mL for serum amyloid A (SAA) and C-reactive protein (CRP), respectively. By comparison, the proposed assay for SAA and CRP was 100-fold and 1000-fold more sensitive than standard colloidal gold strip by using the same antibodies, respectively. This magnetic SERS strip also exhibited excellent stability, specificity, and selectivity for complex samples and could be used as an effective tool for infectious disease diagnostics.

Fast Pathogen Identification Using Single-Cell Matrix-Assisted Laser Desorption/Ionization-Aerosol Time-of-Flight Mass Spectrometry Data and Deep Learning Methods.

In diagnostics of infectious diseases, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) can be applied for the identification of pathogenic microorganisms. However, to achieve a trustworthy identification from MALDI-TOF MS data, a significant amount of biomass should be considered. The bacterial load that potentially occurs in a sample is therefore routinely amplified by culturing, which is a time-consuming procedure. In this paper, we show that culturing can be avoided by conducting MALDI-TOF MS on individual bacterial cells. This results in a more rapid identification of species with an acceptable accuracy. We propose a deep learning architecture to analyze the data and compare its performance with traditional supervised machine learning algorithms. We illustrate our workflow on a large data set that contains bacterial species related to urinary tract infections. Overall we obtain accuracies up to 85% in discriminating five different species.

Identification of Two Pathogens in a Culture‐Negative Prosthetic Joint Infection Using Nanopore Sequencing

Periprosthetic joint infection (PJI) following total joint arthroplasty (TJA) is challenging to diagnose due to lower yield in pathogenic identification (~50%) using conventional microbiologic culture techniques. Here we present a case in which two pathogens were detected using nanopore sequencing from a 50 year‐old male with a suspected, yet culture‐negative, PJI. Three swabs were taken from multiple sites of drainage surrounding the suspected infected right knee joint following a routine arthroplasty (original TKA was done three years prior). DNA was extracted from the swabs using the MasterPure™ Gram Positive DNA Purification Kit (Lucigen). Sample libraries were prepared using the Rapid PCR Barcoding Kit (Oxford Nanopore Technologies) and sequenced on a v9.4.1 MinION flow cell. A total of 1,056,710 reads from all three swabs combined were aligned using a minimum Q score of 7.0 to the RefSeq database on the EPI2ME platform using the “What’s in my pot?” app. A total of 5,540 reads (0.52%) aligned to the bacteria Corynebacterium with the most abundant species being detected as Corynebacterium simulans. A total of 1,886 (0.18%) mapped to the fungi Malassezia globosa. Due to the patient having a visible arthrocutaneous fistula combined with visualized drainage from the joint using a methylene blue challenge test, the prosthetic knee was removed and an antibiotic spacer that contained 6 gm vancomycin, 7.2 gm tobramycin and 100 mg of amphotericin B was implanted. The patient also received a 6 week course of ceftriaxone infusions and has shown rapid clinical improvement. While our sequencing results were not used directly for patient care in this case, it demonstrates the promise that nanopore sequencing has for future use in infectious disease diagnostics.

Clinical Decisions: Detecting Vibriosis in the Modern Era

The accurate identification of Vibrio infections in the clinical microbiology laboratory can have important medical, as well as public health, implications in the detection of fulminant gastroenteritis (cholera) and invasive systemic illnesses, including bacteremia and necrotizing fasciitis. Despite their relatively infrequent occurrence in the United States, an accurate and definitive diagnosis is vital. Due to an ongoing revolution in molecular diagnostics in infectious diseases, which employ techniques such as multiplex PCR and whole-genome sequencing, the field is rapidly changing with respect to the identification of new infectious agents and the use of syndromic-based panels for the detection of enteritis. Despite these advances many issues remain regarding the decision-making process and the detection of vibriosis.

Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification.

Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

Label-Free Biosensors for Laboratory-Based Diagnostics of Infections: Current Achievements and New Trends.

Infections pose a serious global public health problem and are a major cause of premature mortality worldwide. One of the most challenging objectives faced by modern medicine is timely and accurate laboratory-based diagnostics of infectious diseases. Being a key factor of timely initiation and success of treatment, it may potentially provide reduction in incidence of a disease, as well as prevent outbreak and spread of dangerous epidemics. The traditional methods of laboratory-based diagnostics of infectious diseases are quite time- and labor-consuming, require expensive equipment and qualified personnel, which restricts their use in case of limited resources. Over the past six decades, diagnostic technologies based on lateral flow immunoassay (LFIA) have been and remain true alternatives to modern laboratory analyzers and have been successfully used to quickly detect molecular ligands in biosubstrates to diagnose many infectious diseases and septic conditions. These devices are considered as simplified formats of modern biosensors. Recent advances in the development of label-free biosensor technologies have made them promising diagnostic tools that combine rapid pathogen indication, simplicity, user-friendliness, operational efficiency, accuracy, and cost effectiveness, with a trend towards creation of portable platforms. These qualities exceed the generally accepted standards of microbiological and immunological diagnostics and open up a broad range of applications of these analytical systems in clinical practice immediately at the site of medical care (point-of-care concept, POC). A great variety of modern nanoarchitectonics of biosensors are based on the use of a broad range of analytical and constructive strategies and identification of various regulatory and functional molecular markers associated with infectious bacterial pathogens. Resolution of the existing biosensing issues will provide rapid development of diagnostic biotechnologies.

Appropriate Use and Future Directions of Molecular Diagnostic Testing.

Purpose of ReviewMajor technologic advances in two main areas of molecular infectious disease diagnostics have resulted in accelerated adoption or ordering, outpacing implementation, and clinical utility studies. Physicians must understand the limitations to and appropriate utilization of these technologies in order to provide cost-effective and well-informed care for their patients.Recent FindingsRapid molecular testing and, to a lesser degree, clinical metagenomics are now being routinely used in clinical practice. While these tests allow for a breadth of interrogation not possible with conventional microbiology, they pose new challenges for diagnostic and antimicrobial stewardship programs. This review will summarize the most recent literature on these two categories of technologic advances and discuss the few studies that have looked at utilization and stewardship approaches. This review also highlights the future directions for both of these technologies.SummaryThe appropriate utilization of rapid molecular testing and clinical metagenomics has not been well established. More studies are needed to assess their prospective impacts on patient management and antimicrobial stewardship efforts as the future state of infectious disease diagnostics will see continued expansion of these technologic advances.

Development of luminescent serum for diagnostics of animal bacillus anthracix agent

The purpose of the study was to obtain luminescent serum based on highly purified anthracoid globulins to diagnose anthrax agents in animals. To date, there are plenty diagnostic agents that allow rapid and accurate diagnostics of infectious diseases of animals. One of them is the luminescent microscopy of the fluorescent antibody method, which is used as an express method and provides for diagnostics within 3-5 hours. Hyperimmune serum globulins prepared on two types of antigens – protective from strain 55 (VNIIVViM) and capsular from Lange-2 strain at five-fold scheme of introduction of these antigens – were used to make luminescent anthracoid serum. The luminescent serum made on the basis of highly purified anthracoid globulins has a coloring titer (specific activity) of 1:16. When examining the specificity of the obtained luminescent serum in smears from anthrax agent strains, clear fluorescence was observed with more intense luminescence along the periphery of microbial cells.

Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing.

Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.

Antimicrobial susceptibility testing and the Clinician.

The clinicians are confronted with infections in their daily practice, and most of the times they rely on national or international data available to treat these infections. Sometimes it works but sometimes organisms do not respond to antimicrobials due to a variety of factors.
 The data clinician applies is most of the time extrapolated from higher centers, centers of excellence in infectious diseases or he follows guidelines issued by various reputed organizations like WHO, CDC Atlanta, IDSA, NICE, etc.
 Algorithms followed by the medical fraternity in managing infections have been changing with the advent of automation or computerization. Infectious disease diagnostics and management too has been changing fast since the advent of antibiotics. At the same time, these precious molecules are being used inadvertently due to the lack of isolation of organisms.
 A few years back clinicians were perplexed with conflicting outcomes of various microbiologic results from different medical centers that used to rely on age-old diagnostics modalities in microbiology.
 One of the landmarks in modern medicine is the determination of susceptibility of a particular pathogen to select an antimicrobial drug with its adequate blood concentration. In vitro susceptibility of the organism to antibiotics remains a Herculean task. These tests need to properly sampled, stored, incubated, reproduced and accurately performed despite edgy clinician who intends to treat the infection on a war footing.
 The clinicians are confronted with infections in their daily practice, and most of the times they rely on national or international data available to treat these infections. Sometimes it works but sometimes organisms do not respond to antimicrobials due to a variety of factors.
 The data clinician applies is most of the time extrapolated from higher centers, centers of excellence in infectious diseases or he follows guidelines issued by various reputed organizations like WHO, CDC Atlanta, IDSA, NICE, etc.
 Algorithms followed by the medical fraternity in managing infections have been changing with the advent of automation or computerization. Infectious disease diagnostics and management too has been changing fast since the advent of antibiotics. At the same time, these precious molecules are being used inadvertently due to the lack of isolation of organisms.
 A few years back clinicians were perplexed with conflicting outcomes of various microbiologic results from different medical centers that used to rely on age-old diagnostics modalities in microbiology.
 One of the landmarks in modern medicine is the determination of susceptibility of a particular pathogen to select an antimicrobial drug with its adequate blood concentration. In vitro susceptibility of the organism to antibiotics remains a Herculean task. These tests need to properly sampled, stored, incubated, reproduced and accurately performed despite edgy clinician who intends to treat the infection on a war footing.

Detection of Enriched T Cell Epitope Specificity in Full T Cell Receptor Sequence Repertoires.

High-throughput T cell receptor (TCR) sequencing allows the characterization of an individual's TCR repertoire and directly queries their immune state. However, it remains a non-trivial task to couple these sequenced TCRs to their antigenic targets. In this paper, we present a novel strategy to annotate full TCR sequence repertoires with their epitope specificities. The strategy is based on a machine learning algorithm to learn the TCR patterns common to the recognition of a specific epitope. These results are then combined with a statistical analysis to evaluate the occurrence of specific epitope-reactive TCR sequences per epitope in repertoire data. In this manner, we can directly study the capacity of full TCR repertoires to target specific epitopes of the relevant vaccines or pathogens. We demonstrate the usability of this approach on three independent datasets related to vaccine monitoring and infectious disease diagnostics by independently identifying the epitopes that are targeted by the TCR repertoire. The developed method is freely available as a web tool for academic use at tcrex.biodatamining.be.

Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array

BackgroundThe identification and characterization of epitopes facilitate the discovery and development of new therapeutics, vaccines and diagnostics for infectious diseases. In this study, we developed a glutathione S-transferase (GST)-peptide fusion protein microplate array for the identification of linear B-cell epitopes and applied this novel method to the identification of linear B-cell epitopes of SjSP-13, an immunodiagnostic biomarker of schistosomiasis japonica.MethodsSjSP-13 was divided into 17 overlapped peptides (p1-17), and the coding sequence of each peptide was obtained by annealing two complementary oligonucleotides. SjSP-13 peptides were expressed by fusion with an N-terminal GST tag and a C-terminal 6xHis tag. The GST-peptide-His fusion protein was specifically bound to the Immobilizer Glutathione MicroWell 96-well plates without purification. SjSP-13 peptides and core epitopes that could be recognized by sera from schistosomiasis patients were identified by ELISA and confirmed by Western blot analysis. The receiver operating characteristic (ROC) analysis was performed to determine the diagnostic validity of the identified peptide.ResultsFull-length GST-peptide-His fusion proteins were successfully expressed and specifically bound to the Immobilizer Glutathione MicroWell 96-well plates. Two adjacent peptides (p7 and p8) were found to be highly immunogenic in humans. The core epitope of p7 and p8 is an 11-aa peptide (80KCLDVTDNLPE90) and an 8-aa peptide (90EKIIQFAE97), respectively. The area under the ROC curve (AUC) value of the peptide which contains the two identified epitopes is 0.947 ± 0.019. The diagnostic sensitivity and specificity of the peptide is 76.7% (95% CI: 68.8–84.5%) and 100%, respectively.Conclusions90EKIIQFAE97 and 80KCLDVTDNLPE90 are the two linear epitopes of SjSP-13 recognized by patient sera, and could be potential serological markers for schistosomiasis japonica.

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30–45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

Advances in Directly Amplifying Nucleic Acids from Complex Samples.

Advances in nucleic acid amplification technologies have revolutionized diagnostics for systemic, inherited, and infectious diseases. Current assays and platforms, however, often require lengthy experimental procedures and multiple instruments to remove contaminants and inhibitors from clinically-relevant, complex samples. This requirement of sample preparation has been a bottleneck for using nucleic acid amplification tests (NAATs) at the point of care (POC), though advances in “lab-on-chip” platforms that integrate sample preparation and NAATs have made great strides in this space. Alternatively, direct NAATs—techniques that minimize or even bypass sample preparation—present promising strategies for developing POC diagnostic tools for analyzing real-world samples. In this review, we discuss the current status of direct NAATs. Specifically, we surveyed potential testing systems published from 1989 to 2017, and analyzed their performances in terms of robustness, sensitivity, clinical relevance, and suitability for POC diagnostics. We introduce bubble plots to facilitate our analysis, as bubble plots enable effective visualization of the performances of these direct NAATs. Through our review, we hope to initiate an in-depth examination of direct NAATs and their potential for realizing POC diagnostics, and ultimately transformative technologies that can further enhance healthcare.