Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform.

BACKGROUND: With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids. METHODS: Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes. RESULTS: Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease. CONCLUSIONS: The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

A simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings.

BACKGROUND: Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). METHODOLOGY/PRINCIPAL FINDINGS: In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. CONCLUSIONS/SIGNIFICANCE: We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes.

Malaria prevalence defined by microscopy, antigen detection, DNA amplification and total nucleic acid amplification in a malaria-endemic region during the peak malaria transmission season.

OBJECTIVES: To determine the malaria prevalence by microscopy, antigen detection and nucleic acid detection in a defined subpopulation in a Plasmodium falciparum-endemic region during the peak transmission season. METHODS: Blood specimens were collected in a cross-sectional study involving children aged 5-10 years (n = 195) presenting with acute fever to two clinics in Western Kenya. All specimens underwent microscopy, HRP2 and aldolase antigen detection by enzyme immunoassay (EIA), parasite-specific DNA and total nucleic acid (RNA and DNA) by real-time PCR (qPCR) and reverse-transcriptase PCR (qRT-PCR). RESULTS: Microscopy detected 65/195 cases of malaria infection [95% confidence interval (CI) 52-78]. HRP2 and aldolase EIA had similar sensitivity levels detecting antigen in 65/195 (95% CI, 52-78) and 57/195 (95% CI, 45-70) cases. Discordants in antigen detection vs. microscopy occurred at <470 parasites/µl and <4900 parasites/µl for HRP2 and aldolase, respectively. Detection of total nucleic acid allowed a 3 log lower limit of detection than just DNA detection by real-time PCR in vitro. In clinical specimens, 114/195 (95% CI, 100-127) were qPCR positive (DNA), and 187/195 (95% CI, 179-191) were qRT-PCR positive (DNA plus RNA). CONCLUSIONS: The prevalence of submicroscopic malaria infection was significantly higher when detecting total nucleic acid than just DNA in this outpatient population during the high transmission season. Defining standards for submicroscopic infection will be important for control programmes, diagnostics development efforts and molecular epidemiology studies.

Non-instrumented nucleic acid amplification (NINA): instrument-free molecular malaria diagnostics for low-resource settings.

We have achieved the first complete, non-instrumented nucleic acid amplification test (NAAT) using a calcium oxide heat source thermally linked to an engineered phase change material. These two components alone maintain a thermal profile suitable for the loop-mediated isothermal amplification assay. Starting with computational fluid dynamics analysis, we identified nominal geometry for the exothermic reaction chamber, phase change material chamber, thermal insulation, and packaging. Using this model, we designed and fabricated an alpha prototype assay platform. We have verified the function of this multi-pathogen-capable platform with both fluorescent and visual turbidity indications using samples spiked with malaria DNA. Both the exothermically heated platform samples and samples heated on a Perkin-Elmer GeneAmp9600 thermocycler were first incubated at 62°C for 45 minutes, then heated to 95°C to terminate enzyme activity, then analyzed. Results from the exothermically heated, non-instrumented platform were comparable to those from the thermocycler. These developments will enable point-of-care diagnostics using accurate NAATs which until now have required a well-equipped laboratory. The aim of this research is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where assays such as immunochromatographic strip tests are successfully used but where there is no access to the infrastructure and logistics required to operate and maintain instrument-based diagnostics.

Cost analysis of centralized viral load testing for antiretroviral therapy monitoring in Nicaragua, a low-HIV prevalence, low-resource setting.

BACKGROUND: HIV viral load testing as a component of antiretroviral therapy monitoring is costly. Understanding the full costs and the major sources of inefficiency associated with viral load testing is critical for optimizing the systems and technologies that support the testing process. The objective of our study was to estimate the costs associated with viral load testing performed for antiretroviral therapy monitoring to both patients and the public healthcare system in a low-HIV prevalence, low-resource country. METHODS: A detailed cost analysis was performed to understand the costs involved in each step of performing a viral load test in Nicaragua, from initial specimen collection to communication of the test results to each patient's healthcare provider. Data were compiled and cross referenced from multiple information sources: laboratory records, regional surveillance centre records, and scheduled interviews with the key healthcare providers responsible for HIV patient care in five regions of the country. RESULTS: The total average cost of performing a viral load test in Nicaragua varied by region, ranging from US$99.01 to US$124.58, the majority of which was at the laboratory level: $88.73 to $97.15 per specimen, depending on batch size. The average cost to clinics at which specimens were collected ranged from $3.31 to $20.92, depending on the region. The average cost per patient for transportation, food, lodging and lost income ranged from $3.70 to $14.93. CONCLUSIONS: The quantitative viral load test remains the single most expensive component of the process. For the patient, the distance of his or her residence from the specimen collection site is a large determinant of cost. Importantly, the efficiency of results reporting has a large impact on the cost per result delivered to the clinician and utility of the result for patient monitoring. Detailed cost analysis can identify opportunities for removing barriers to effective antiretroviral therapy monitoring programmes in limited-resource countries with low HIV prevalence.

Outpatient upper respiratory tract viral infections in children with malaria symptoms in Western Kenya.

A cross-sectional study was performed in children 5 through 10 years of age presenting to outpatient clinics in Nyanza Province, Kenya, in which nasal swab and blood specimens were collected during the high malaria transmission season. Patients presenting with malaria-like symptoms within 4 days of fever onset were enrolled in the study. Plasmodium parasitemia was determined by blood smear microscopy. Nasal swabs were screened for a panel of respiratory viruses by polymerase chain reaction. Influenza A, rhinoviruses, and other respiratory viruses were detected in 18%, 26%, and 12% of 197 specimens, respectively. Four of 36 patients with influenza A had a positive malaria blood slide, compared with 20 of 52 patients with rhinovirus. A significant burden of disease caused by influenza A in febrile children during the study period was observed, highlighting the need for further research into the burden of influenza disease in regions where malaria is holoendemic.

Laboratory operations, specimen processing, and handling for viral load testing and surveillance.

RNA remains the most informative and accurate biomarker for human immunodeficiency virus type 1 load diagnostics and for surveillance of drug resistance markers. Viral load testing by nucleic acid amplification currently is a complex and expensive test that is restricted to centralized laboratory testing. Successful extension of centralized viral load testing to rural or remote settings is a major challenge. Emerging nucleic acid-based technologies are progressing rapidly toward platforms appropriate for field use in low-resource settings, leaving a growing gap for sample processing technologies that complement them. One area in which new technologies could be applied to improve access is clinical specimen preservation and processing. Novel technologies that extract nucleic acid from clinical specimens and stabilize it at the point of specimen collection could fill this gap. In addition, these technologies may provide alternative viral load detection and surveillance solutions to the current centralized laboratory testing paradigm.

Towards non- and minimally instrumented, microfluidics-based diagnostic devices.

In many health care settings, it is uneconomical, impractical, or unaffordable to maintain and access a fully equipped diagnostics laboratory. Examples include home health care, developing-country health care, and emergency situations in which first responders are dealing with pandemics or biowarfare agent release. In those settings, fully disposable diagnostic devices that require no instrument support, reagent, or significant training are well suited. Although the only such technology to have found widespread adoption so far is the immunochromatographic rapid assay strip test, microfluidics holds promise to expand the range of assay technologies that can be performed in formats similar to that of a strip test. In this paper, we review progress toward development of disposable, low-cost, easy-to-use microfluidics-based diagnostics that require no instrument at all. We also present examples of microfluidic functional elements--including mixers, separators, and detectors--as well as complete microfluidic devices that function entirely without any moving parts and external power sources.

A small molecule-kinase interaction map for clinical kinase inhibitors.

Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.