Development of a Decision Aid for Patients and Families Considering Hospice.

Background: Hospice is underutilized. Miscommunication, decisional complexity, and misunderstanding around engaging hospice may contribute. Shared decision making (SDM), aided by patient decision aids (PtDAs), can improve knowledge and decision quality. Currently, there are no freely available hospice-specific PtDA to facilitate conversions between patients and providers about hospice care. Objective: To develop a theory-based and unbiased hospice specific PtDA. Design: Guided by the Ottawa Decision Support Framework and International Patient Decision Aid Standards, we used a theory-driven, eight-step, iterative, user-centered approach with multistakeholder input to develop a hospice-specific PtDA for anyone facing end-of-life decisions. Subjects: Feedback was obtained from a 10-member Patient Advisory Panel composed of lay patient advisors; focus groups of hospice providers, family caregivers, and patients; and the Palliative Care Research Group at University of Colorado Hospital consisting of palliative care physicians, midlevel providers, nurses, social workers, chaplains, and researchers. Results: There are many challenges in developing an unbiased hospice decision aid, including (1) balancing the provision of education (eligibility, payment) with decisional support, (2) clarifying values and incorporating emotion, (3) ideally representing the potential downsides of hospice, and (4) adequately capturing and describing care alternatives to hospice. Within this context, we developed a 12-page article and 17-minute video PtDAs. The PtDA openly acknowledges the emotional complexity of the decision and incorporates values clarification techniques to help decision makers reflect and evaluate their goals and preferences for end-of-life care. Conclusions: Hospice decision making is complex and emotional, demanding high-quality SDM aided by a formal PtDA. This work resulted in a freely available article and video PtDA for patients considering hospice. The effectiveness and implementation of these tools will be studied in future research. Clinical Trials Registration (NCT03794700 & NCT04458090).

An Electronically Delivered Patient-Activation Tool for Intensification of Medications for Chronic Heart Failure With Reduced Ejection Fraction: The EPIC-HF Trial.

BACKGROUND: Major gaps exist in the routine initiation and dose up-titration of guideline-directed medical therapies (GDMT) for patients with heart failure with reduced ejection fraction. Without novel approaches to improve prescribing, the cumulative benefits of heart failure with reduced ejection fraction treatment will be largely unrealized. Direct-to-consumer marketing and shared decision making reflect a culture where patients are increasingly involved in treatment choices, creating opportunities for prescribing interventions that engage patients. METHODS: The EPIC-HF (Electronically Delivered, Patient-Activation Tool for Intensification of Medications for Chronic Heart Failure with Reduced Ejection Fraction) trial randomized patients with heart failure with reduced ejection fraction from a diverse health system to usual care versus patient activation tools-a 3-minute video and 1-page checklist-delivered electronically 1 week before, 3 days before, and 24 hours before a cardiology clinic visit. The tools encouraged patients to work collaboratively with their clinicians to "make one positive change" in heart failure with reduced ejection fraction prescribing. The primary endpoint was the percentage of patients with GDMT medication initiations and dose intensifications from immediately preceding the cardiology clinic visit to 30 days after, compared with usual care during the same period. RESULTS: EPIC-HF enrolled 306 patients, 290 of whom attended a clinic visit during the study period: 145 were sent the patient activation tools and 145 were controls. The median age of patients was 65 years; 29% were female, 11% were Black, 7% were Hispanic, and the median ejection fraction was 32%. Preclinic data revealed significant GDMT opportunities, with no patients on target doses of ß-blocker, sacubitril/valsartan, and mineralocorticoid receptor antagonists. From immediately preceding the cardiology clinic visit to 30 days after, 49.0% in the intervention and 29.7% in the control experienced an initiation or intensification of their GDMT (P=0.001). The majority of these changes were made at the clinician encounter itself and involved dose uptitrations. There were no deaths and no significant differences in hospitalization or emergency department visits at 30 days between groups. CONCLUSIONS: A patient activation tool delivered electronically before a cardiology clinic visit improved clinician intensification of GDMT. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03334188.

Show Me the Money: Patients' Perspectives on a Decision Aid for Sacubitril/Valsartan Addressing Out-of-Pocket Cost.

BACKGROUND: Out-of-pocket medication costs for patients who have heart failure with reduced ejection fraction may be an important part of shared decision-making, but cost has generally been excluded from clinical discussions. This study reports patients' perspectives on a decision aid for sacubitril/valsartan that explicitly addresses out-of-pocket costs. METHODS: Structured, in-depth interviews were conducted with 20 patients with heart failure with reduced ejection fraction from 2 medical centers to elicit their views on a publicly available decision aid for sacubitril/valsartan that explicitly incorporates considerations related to out-of-pocket costs. Qualitative descriptive analysis was conducted. RESULTS: Key themes identified were general enthusiasm for decision aids for medication decisions, openness on the part of patients to incorporation of cost into decision-making and the decision aid, requests for greater specificity regarding patient-specific cost, and challenges communicating evidence of benefit in a way that allows patients to make cost-benefit analyses for themselves. Patients also raised questions regarding logistical challenges of incorporating a decision aid into the normal clinical and decision-making workflow. CONCLUSIONS: Patients were receptive to the inclusion of out-of-pocket cost as relevant in a decision aid for sacubitril/valsartan. Key challenges to effective integration of cost in these decisions include developing mechanisms for acquiring reliable patient-specific cost estimates and addressing patients' difficulties (and sometimes skepticism) applying trial evidence to their own situation. In addition, implementation strategies are important to develop to facilitate decision aid integration for routine medical decisions into clinic workflow.

An Electronically delivered, Patient-activation tool for Intensification of medications for Chronic Heart Failure with reduced ejection fraction: Rationale and design of the EPIC-HF trial.

BACKGROUND: Heart failure with reduced ejection fraction (HFrEF) benefits from initiation and intensification of multiple pharmacotherapies. Unfortunately, there are major gaps in the routine use of these drugs. Without novel approaches to improve prescribing, the cumulative benefits of HFrEF treatment will be largely unrealized. Direct-to-consumer marketing and shared decision making reflect a culture where patients are increasingly involved in treatment choices, creating opportunities for prescribing interventions that engage patients. HYPOTHESIS: Encouraging patients to engage providers in HFrEF prescribing decisions will improve the use of guideline-directed medical therapies. DESIGN: The Electronically delivered, Patient-activation tool for Intensification of Chronic medications for Heart Failure with reduced ejection fraction (EPIC-HF) trial randomizes patients with HFrEF to usual care versus patient-activation tools-a 3-minute video and 1-page checklist-delivered prior to cardiology clinic visits that encourage patients to work collaboratively with their clinicians to intensify HFrEF prescribing. The study assesses the effectiveness of the EPIC-HF intervention to improve guideline-directed medical therapy in the month after its delivery while using an implementation design to also understand the reach, adoption, implementation, and maintenance of this approach within the context of real-world care delivery. Study enrollment was completed in January 2020, with a total 305 patients. Baseline data revealed significant opportunities, with <1% of patients on optimal HFrEF medical therapy. SUMMARY: The EPIC-HF trial assesses the implementation, effectiveness, and safety of patient engagement in HFrEF prescribing decisions. If successful, the tool can be easily disseminated and may inform similar interventions for other chronic conditions.

Sildenafil Administration in Dogs Heterozygous for a Functional Null Mutation in Pde6a: Suppressed Rod-Mediated ERG Responses and Apparent Retinal Outer Nuclear Layer Thinning.

This study was designed to assess risk for retinal toxicity associated with administration of high-dose sildenafil citrate to dogs heterozygous for a functionally null mutation in Pde6a over a 4-month period. Three Pde6a +/- dogs were administered 14.3 mg/kg sildenafil per os and two Pde6a +/- dogs placebo once daily for 16 weeks. Three Pde6a +/+ dogs were administered sildenafil for 7 days. Ophthalmic examination, vision testing, and electroretinography (ERG) were regularly performed. At study termination, dogs were euthanized and globes collected. Retinal layer thickness and photoreceptor nuclei counts were determined from plastic sections. In both Pde6a +/- and Pde6a +/+ sildenafil-treated (ST) dogs, elevation of dark-adapted b-wave threshold and unmasking of the scotopic threshold response (STR) were observed. Sildenafil treated Pde6a +/- dogs had significantly thinner ONL (24.90 +/-1.88 µm, p = 0.004) and lower photoreceptor nuclei counts (273.6 +/- 29.3 cells/100 µm, p = 0.008) compared to measurements (35.90 +/- 1.63 µm) and counts (391.5 +/-27.0 cells/100 µm) from archived untreated Pde6a +/- dogs.

An association between systemic cyclosporine administration and development of acute bullous keratopathy in cats.

OBJECTIVE: To determine whether any association exists between the onset of feline acute bullous keratopathy (ABK) and administration of systemic corticosteroid or immunosuppressive therapy. ANIMALS STUDIED: Medical records of cats diagnosed with ABK between the years of 2000 and 2008 were retrospectively reviewed. Breed, age at diagnosis, weight, systemic disease status, eye affected, ophthalmic examination findings, systemic and topical therapy instituted, dosage and duration of therapy, visual outcome and histopathological analyses were recorded in cases meeting the inclusion criteria. RESULTS: A total of 12 cats of a surveyed population of 70 167 met the inclusion criteria with 17/24 eyes affected by ABK. Medical and/or surgical therapy was utilized for management of ABK with 13/17 eyes remaining sighted at the time of last follow-up. In a subset of cases corneal cytology, aerobic bacterial culture, FHV-1 PCR, virus isolation and/or histopathology were performed; no infectious organisms were identified. A rupture in Descemet's membrane of the cornea was identified histologically in two globes. A total of 10 of 12 cats had been previously diagnosed with ongoing systemic disease. A total of 10 of 12 cats were receiving systemic therapy, and a significant association (P < 0.001) was noted between systemic administration of corticosteroids and/or cyclosporine A and the development of ABK. A total of 8 of 10 cats were administered oral prednisolone at doses between 1-2 mg/kg every 12-24 h. A total of 5 of 8 cats receiving oral prednisolone were concurrently administered oral cyclosporine at doses of 1.5-7 mg/kg every 12-24 h. Systemic cyclosporine therapy was found to be a significant risk factor (P < 0.001) for ABK development, while systemic prednisolone was not significant (P = 0.10). CONCLUSIONS: Systemic cyclosporine administration appears to be a risk factor for development of ABK in the population of cats studied.

Low-concentration initiator primers improve the amplification of gene targets with high sequence variability.

The amplification and detection of diverse strains of an infectious virus or bacteria, or variants within a gene family is important for both clinical and basic research but can be difficult using conventional PCR. This report describes and illustrates a novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family. Different subgroups of CTX-M genes exhibit low sequence identity, but accurate and efficient detection of these variants is critical because they all confer resistance to penicillin, cefotaxime, and other antibiotics of the beta-lactam class. The method combines a single pair of "thermodynamic consensus primers" (tcPrimers) with one or more "initiator primers" (iPrimers), added at low concentration (5-10 nM). Each iPrimer improves the initial amplification of one or more variants because it has fewer mismatches to its intended target than the more abundant tcPrimers. As a result of initial amplification, each heterogeneous sequence is shifted stepwise toward a better match with the tcPrimers. As soon as the tcPrimer hybridization takes place, amplification proceeds with high efficiency. The tcPrimer pairs can be designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR. LATE-PCR offers the advantage of generating single-stranded DNA that can be characterized for different gene variants in the same closed tube, using low-temperature mismatch-tolerant fluorescent probes.

Rapid detection and identification of hepatitis C virus (HCV) sequences using mismatch-tolerant hybridization probes: A general method for analysis of sequence variation.

Detection and identification of highly variable viral sequences is important for tracking infectious outbreaks and determining treatment regimens using targeted drug therapy. This report describes a single tube assay that is able to distinguish extensive sequence variation in hepatitis C virus (HCV) by using mismatch tolerant probes to analyze single-stranded amplicons generated with reverse transcription linear-after-the-exponential PCR (RT-LATE-PCR). Detection and identification of sequences from the 5' non-coding region (NCR) of 31 different HCV strains was first evaluated via hybridization of two fluorescently labeled, mismatch-tolerant probes to synthetic DNA strands. The resulting data were used to calculate the ratio of fluorescent signals for the two probes over a wide temperature range as well as the melting temperature (Tm) of each probe with the targets. Although the Tm measurements alone distinguished only 5 sequences from the others, fluorescent signal ratio analysis provided a unique set of values for 27 of the 31 strains. RT-LATE-PCR was then used to amplify Armored RNA (AR) containing the 5' NCR of five different strains of HCV. Melting analysis of the resulting single-stranded DNA with the two probes distinguished all five AR sequences. This assay can be expanded to include additional gene segments, and it points the way to construction of highly informative single-tube assays for HCV and other RNA viruses.

Rapid detection of TEM-type extended-spectrum ß-lactamase (ESBL) mutations using lights-on/lights-off probes with single-stranded DNA amplification.

Rapid identification of specific TEM-type ß-lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the blaTEM gene, as well as detecting widely spaced extended-spectrum ß-lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25°C to 75°C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.

Rapid detection of sequence variation in Clostridium difficile genes using LATE-PCR with multiple mismatch-tolerant hybridization probes.

A novel molecular assay for Clostridium difficile was developed using Linear-After-The-Exponential polymerase chain reaction (LATE-PCR). Single-stranded DNA products generated by LATE-PCR were detected and distinguished by hybridization to fluorescent mismatch-tolerant probes, as the temperature was lowered after amplification in 5(°)C intervals between 65°C and 25°C. Single-tube multiplex reactions for tcdA, tcdB, tcdC, and cdtB (binary toxin) sequences were initially optimized using synthetic targets and were subsequently done using genomic DNA; each target was detected and characterized by hybridization to one or more probes of a different fluorescent color. In the case of tcdC, three probes, each labeled with a Quasar fluorophore, hybridize to different locations with known mutations, including the deletion at nucleotide 117 in ribotype 027 strains and the premature stop codon mutation at nucleotide 184 in ribotype 078 strains, each of which is associated with hypervirulent infections. These and other tcdC mutations were distinguished from the reference sequence, as well as from each other by changes in the fluorescent contour generated from the combined Quasar-labeled probes. Specific variations in tcdA and tcdB were also identified in the multiplex assay, including those that identified strains lacking toxin A production. This single closed-tube assay generates substantially more information about virulent C. difficile than currently available commercial assays and could be further expanded to provide strain typing.

Bilateral uveal metastasis of a subcutaneous fibrosarcoma in a cat.

A 6-year-old neutered male domestic short-haired cat was presented to the Comparative Ophthalmology service at Michigan State University with a 3-week history of decreased appetite and redness of the left eye. The left forelimb had been removed 15 months previously because of the presence of a subcutaneous fibrosarcoma. In the left globe, a large iridal mass was associated with increased intraocular pressure and retinal detachment. A smaller mass involving the right iris was also present. Imaging revealed a 2-cm mass in the left caudodorsal lung lobe, and abdominal ultrasound showed multifocal bilateral renal masses. Aspirates of these masses were nondiagnostic. The left globe was removed for palliative reasons, and histopathology showed that fibrosarcoma was infiltrating the iris, choroid, and optic nerve. Despite systemic chemotherapy with doxorubicin, the animal died 4 months after initial presentation. Histopathology confirmed highly angioinvasive metastatic fibrosarcoma also in the right uveal tract, the lungs, and both kidneys.

LATE-PCR and allied technologies: real-time detection strategies for rapid, reliable diagnosis from single cells.

Accurate detection of gene sequences in single cells is the ultimate challenge of PCR sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of Linear-After-The-Exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at -different concentrations, but with novel design criteria to insure high efficiency and specificity. LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single-cell genetic diagnosis.

Safe and effective treatment of early suprahepatic inferior vena caval outflow compromise following orthotopic liver transplantation using percutaneous transluminal angioplasty and stent placement.

PURPOSE: To describe the safety and efficacy of percutaneous transluminal angioplasty and stent placement in patients presenting with suprahepatic inferior vena cava (IVC) outflow compromise in the early postoperative period following orthotopic liver transplantation. METHODS AND RESULTS: Between October 2002 and April 2009, 3 patients presented with IVC outflow compromise in the first 2 months following orthotopic liver transplantation. All 3 underwent percutaneous transluminal angioplasty and stent placement without complication and showed significant clinical improvement at short and intermediate term follow-up. CONCLUSION: Percutaneous transluminal angioplasty and Gianturco stent placement is a safe and effective treatment for IVC outflow compromise in the early postoperative period following orthotopic liver transplantation.

Pan-serotypic detection of foot-and-mouth disease virus by RT linear-after-the-exponential PCR.

A reverse transcription Linear-After-The-Exponential polymerase chain reaction (RT LATE-PCR) assay was evaluated for detection of foot-and-mouth disease virus (FMDV). This pan-serotypic assay targets highly conserved sequences within the 3D (RNA polymerase) region of the FMDV genome, and uses end-point hybridisation analysis of a single mismatch-tolerant low temperature probe to confirm the identity of the amplicons. An Armored RNA served as an internal control to validate virus negative results. The ability of the assay to identify FMDV was directly compared to a real-time RT-PCR assay routinely used by reference laboratories. The analytical sensitivity of the RT LATE-PCR assay was 10 genomic copies and the dynamic range of the test was identical to real-time RT-PCR based on decimal dilutions of an FMDV-positive sample. This pan-serotypic assay was able to detect FMDV in a broad range of clinical samples collected from field cases of FMD (n = 121), while samples of other viruses causing vesicular disease in livestock and genetic relatives of FMDV were negative. In addition to the laboratory-based utility of this diagnostic test, the RT LATE-PCR assay format has potential application for use in a portable ("point-of-care") device designed to achieve rapid detection of FMDV in the field.

Dislocation of the knee: imaging findings.

Dislocations of the knee are relatively uncommon injuries. However, the incidence of this injury appears to be increasing. Knee dislocations are most often high velocity blunt injuries, with motor vehicle accidents being a frequent etiology. Other causes include falls from height, athletic injuries, farming and industrial accidents, and even low velocity mechanisms such as a misstep into a hole. Likewise, minor trauma in the morbidly obese is increasingly recognized as a mechanism of knee dislocation. Multiple forms of dislocation exist, with the common factor being disruption of the tibiofemoral articulation. Dislocation can occur in a variety of directions depending on the mechanism of injury. The most common dislocation is anterior, which may be seen in hyperextension injuries such as martial arts kicking. The "dashboard injury" of motor vehicle accidents can result in a posterior dislocation of the knee. Lateral and rotary dislocations are less common. Knee dislocation is more commonly diagnosed in men, with a mean age of 23 to 31 years old. This is the very patient population encountered by Special Operations Forces (SOF) healthcare providers. Given the mechanisms of injury noted above, it is reasonable to conclude that knee dislocations may be seen in a young, active SOF patient population, particularly those engaged in parachuting, fast-roping/rappelling, driving at high speeds during military operations, and mixed martial arts.

Development of an implantable D-serine biosensor for in vivo monitoring using mammalian D-amino acid oxidase on a poly (o-phenylenediamine) and Nafion-modified platinum-iridium disk electrode.

D-serine has been implicated as a brain messenger, promoting not only neuronal signalling but also synaptic plasticity. Thus, a sensitive tool for D-serine monitoring in brain is required to understand the mechanisms of D-serine release from glia cells. A biosensor for direct fixed potential amperometric monitoring of D-serine incorporating mammalian D-amino acid oxidase (DAAO) immobilized on a Nafion coated poly-ortho-phenylenediamine (PPD) modified Pt-Ir disk electrode was therefore developed. The combined layers of PPD and Nafion enhanced the enzyme activity and biosensor efficiency by approximately 2-fold compared with each individual layer. A steady state response time (t(90%)) of 0.7+/-0.1s (n=8) and limit of detection 20+/-1 nM (n=8) were obtained. Cylindrical geometry showed lower sensitivity compared to disk geometry (61+/-7 microA cm(-2) mM(-1), (n=4), R(2)=0.999). Interference by ascorbic acid (AA), the main interference species in the central nervous system and other neurochemical electroactive molecules was negligible. Implantation of the electrode and microinjection of D-serine into rat brain striatal extracellular fluid demonstrated that the electrode was capable of detecting D-serine in brain tissue in vivo.

Primary angiosarcoma of the abdominal aorta: a case report and literature review (aortic angiosarcoma).

Fewer than 140 cases of aortic sarcoma have been reported with only 34 classified as angiosarcoma. These rare malignancies most often mimic aortoiliac occlusive or aneurysmal arteriosclerotic disease both clinically and on imaging studies, and the large majority are unexpected and diagnosed pathologically on a surgical specimen or at autopsy. A 42-year-old woman who presented with low back pain and claudication was shown by CT and angiography to have infrarenal aortic occlusion. Angiosarcoma was an unexpected pathologic diagnosis based on tissue removed during aortobifemoral bypass surgery. Pre and postoperative CT and angiography are presented along with a review of the literature.

Treatment of hemodialysis graft pseudoaneurysms with stent grafts: institutional experience and review of the literature.

Hemodialysis access grafts are an important component of the treatment of patients with renal failure. Because access sites are limited, maximizing graft lifespan is of major importance to dialysis patients. Pseudoaneurysm formation is a rare, but important complication potentially limiting the longevity of dialysis grafts. With rapidly advancing technology, placement of stent grafts in patients with end-stage renal disease is an important step in prolonging the life of the graft. We conducted a review of the literature regarding stent-graft use for hemodialysis access. In addition, we looked at our experience utilizing the Viabahn(®) (W. L. Gore & Associates, Newark, DE) stent graft in pseudoaneurysm repair. Our patients achieved primary patency of their grafts for 1, 5, and 9 months, respectively. No complications related to stent-graft implementation have been encountered in six stent-graft implants over the course of 29 months.

Role of interventional radiology in the management of complications after pancreaticoduodenectomy.

BACKGROUND: This study evaluated the role of interventional radiology (IR) procedures to manage complications after pancreaticoduodenectomy. METHODS: A retrospective review was made of the records of patients with postsurgical complications managed with IR. RESULTS: Among the 440 patients reviewed, the mortality, morbidity and reoperation rates were 1.6%, 36%, and 2%, respectively. Complications occurred in 159 patients, of which 39 (25%) required > or = 1 IR procedures. Of those 39 patients, 72% underwent percutaneous drainage of an intra-abdominal abscess, 18% underwent percutaneous biliary drainage, and 10% underwent angiography for gastrointestinal bleeding or pseudoaneurysm. The reoperation rate among the 159 patients with complications was 6% (n = 9). Reoperation was avoided in 90% of patients receiving IR. Four patients underwent reoperation despite IR for persistent abscess, pancreatic fistula, anastomotic disruption, or mesenteric venous bleeding. CONCLUSIONS: The majority of complications occurring after pancreaticoduodenectomy can be managed effectively using IR, thus minimizing morbidity and the need for reoperation.

Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing.

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.