Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing.

BACKGROUND: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker. METHODS: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (TB) patients with minimal bias using next-generation sequencing (NGS). A combination of DNA extraction and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments was employed. Urine cfDNA from 10 HIV-positive patients with pulmonary TB and two MTB-negative controls was examined. RESULTS: MTB-derived cfDNA was identifiable by NGS from all MTB-positive patients and was absent from negative controls. MTB cfDNA was significantly shorter than human cfDNA, with median fragment lengths of ≤19-52 bp and 42-92 bp, respectively. MTB cfDNA abundance increased exponentially with decreased fragment length, having a peak fragment length of ≤19 bp in most samples. In addition, we identified a larger fraction of short human genomic cfDNA, ranging from 29 to 53 bp, than previously reported. Urine cfDNA fragments spanned the MTB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately over-represented. CONCLUSIONS: TB urine cfDNA is a potentially powerful biomarker but is highly fragmented, necessitating special procedures to maximize its recovery and detection.

Evaluating extraction methods to study canine urine microbiota.

The urinary microbiota is the collection of microbes present in urine that may play a role in host health. Studies of urine microbiota have traditionally relied upon culturing methods aimed at identifying pathogens. However, recent culture-free sequencing studies of the urine microbiota have determined that a diverse array of microbes is present in health and disease. To study these microbes and their potential role in diseases like bladder cancer or interstitial cystitis, consistent extraction and detection of bacterial DNA from urine is critical. However, urine is a low biomass substrate, requiring sensitive methods to capture DNA and making the risk of contamination high. To address this challenge, we collected urine samples from ten healthy dogs and extracted DNA from each sample using five different commercially available extraction methods. Extraction methods were compared based on total and bacterial DNA concentrations and bacterial community composition and diversity assessed through 16S rRNA gene sequencing. Significant differences in the urinary microbiota were observed by dog and sex but not extraction method. The Bacteremia Kit yielded the highest total DNA concentrations (Kruskal-Wallis, p = 0.165, not significant) and the highest bacterial DNA concentrations (Kruskal-Wallis, p = 0.044). Bacteremia also extracted bacterial DNA from the greatest number of samples. Taken together, these results suggest that the Bacteremia kit is an effective option for studying the urine microbiota. This work lays the foundation to study the urine microbiome in a wide range of urogenital diseases in dogs and other species.

Benchmarking DNA isolation kits used in analyses of the urinary microbiome.

The urinary microbiome has been increasingly characterized using next-generation sequencing. However, many of the technical methods have not yet been specifically optimized for urine. We sought to compare the performance of several DNA isolation kits used in urinary microbiome studies. A total of 11 voided urine samples and one buffer control were divided into 5 equal aliquots and processed in parallel using five commercial DNA isolation kits. DNA was quantified and the V4 segment of the 16S rRNA gene was sequenced. Data were processed to identify the microbial composition and to assess alpha and beta diversity of the samples. Tested DNA isolation kits result in significantly different DNA yields from urine samples. DNA extracted with the Qiagen Biostic Bacteremia and DNeasy Blood & Tissue kits showed the fewest technical issues in downstream analyses, with the DNeasy Blood & Tissue kit also demonstrating the highest DNA yield. Nevertheless, all five kits provided good quality DNA for high throughput sequencing with non-significant differences in the number of reads recovered, alpha, or beta diversity.

Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples.

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6-117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25-150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA.

BACKGROUND: Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom's MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom's MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom's MLST scheme in clinical samples with low concentrations of Chlamydia DNA. RESULTS: In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom's MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. CONCLUSIONS: The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

Serological survey and DNA screening of Leptospira spp. in free-living adult tufted capuchin monkeys (Cebus apella nigritus) in a forest reserve Southeast São Paulo State, Brazil.

BACKGROUND: Leptospirosis is an important anthropozoonosis. The study investigated the presence of anti-Leptospira antibodies and detection of Leptospira spp DNA in the urine as well as the biochemical profile in Neotropical wild primates living in a forest reserve from Southeast São Paulo State, Brazil. METHODS: Blood samples were obtained from 50 adult tufted capuchin monkeys (Cebus apella nigritus). Urine samples were obtained only from male primates. The screening for antibodies against Leptospira spp was evaluated by microscopic agglutination test (MAT). Leptospira DNA in the urine was evaluated by polymerase chain reaction (PCR) considering the target gene LipL32. Biochemical profile was evaluated by using a spectrophotometer. RESULTS: The MAT results included 39 (78%) serum reactive animals with the proportions of 28/39 males and 11/39 females. The most frequent reactive serogroups were Icterohemorrhagiae, Canicola, and Autumnalis. All urine samples were negative for leptospiral DNA. There were no significant differences between sexes for aspartate aminotransferase (AST) and alkaline phosphatase values, but alanine aminotransferase (ALT), creatinine, glucose, and urea were significantly higher in males. CONCLUSIONS: Tufted capuchin monkeys were sera reactive against leptospirosis. Prevalence was similar for the 2 sexes. Leptospiral DNA was not detected in the urine of sera reactive primates tested by the MAT method. ALT, creatinine, glucose, and urea values were higher in male animals.

Development of lipL32 real-time PCR combined with an internal and extraction control for pathogenic Leptospira detection.

At least two real-time PCRs for the early diagnosis of leptospirosis have been described, evaluated and validated. However, at least one other report suggested adaptation and modification of primers and probes used in these assays since additional Leptospira species have been described and the primers and probe in use possess a serious mismatch to corresponding target sequence. In this study we developed a real-time PCR for detection of pathogenic Leptospira based on the lipL32 gene. The present method consists of generic primers and probes based on target sequence of 10 pathogenic Leptospira species including Leptospira interrogans. The hybridization, annealing and extension temperature (60°C) were optimized as the optimal temperature of the DNA polymerase enzyme which is used in the amplification reaction. The present assay has a high analytical sensitivity and specificity; the calculated diagnostic sensitivity and specificity were 93.0% and 98.3% respectively. Moreover, the present method includes an internal control which enables easy detection of false negative results and an optional extraction control which enables the estimation of the DNA extraction efficiency.

Usefulness of Nested Polymerase Chain Reaction with Clinical Specimens for Diagnosis of Leptospirosis: a Case Series and a Review of Literature.

A culture of the Leptospira species and the microscopic agglutination test (MAT) are considered as the reference standard for the diagnosis of leptospirosis, but both tests are imperfect for early diagnosis. We describe 4 patients diagnosed with leptospirosis using nested polymerase chain reaction (N-PCR) that targeted the 16S rRNA gene and the passive hemagglutination assay (PHA). In our 4 cases, Leptospira DNA in the urine, plasma, or cerebrospinal fluid (CSF), was detected by N-PCR in the early phase of leptospirosis, except in the sample from the buffy coat. Especially, case 3 showed that N-PCR with the urine and CSF was positive 8 days after symptom onset, but not for the plasma or buffy coat. We report 4 cases of leptospirosis that were diagnosed by N-PCR that targeted the 16S rRNA gene with urine, plasma, or CSF, but not the buffy coat. Three were cured by doxycycline but the case 4 was fatal. Detection of Leptospira DNA by PCR from the urine and CSF, in addition to plasma, may be helpful to confirm the diagnosis.

Successful Leptospira genotyping strategy on DNA extracted from canine biological samples.

Leptospirosis is an emerging worldwide zoonosis with a changing epidemiology responsible for an acute disease in humans and dogs. A better knowledge of the responsible bacterium Leptospira and in particular its various serovars and serogroups prevalence is essential for better diagnosis and prevention of the disease. The gold standard for leptospirosis diagnosis is the Microscopic Agglutination Test (MAT) but it requires long and fastidious laboratory work and sometimes results in controversial data. For these reasons, PCR-based techniques for detection of pathogenic leptospiral DNA in biological samples are currently replacing the MAT. However, these strategies do not provide any information regarding the infecting serovar or serogroup. In this study, an optimized genotyping method is described to allow the identification of Leptospira ssp. directly at serovars level using DNA extracted from canine blood and urine. 16S rDNA, Variable Number Tandem Repeat (VNTR) and Multispacer Sequence Typing (MST) protocols were adapted to biological samples. Eighty-eight DNA samples were analyzed from 72 different European canine clinical cases of leptospirosis confirmed by real-time PCR. 92% of DNA samples with Ct values below 34 were fully typed, and typing success decreased to about 30% for the other samples. Typing failure also showed a specie-specific correlation, with 63% of complete typing for L. interrogans and only 40% for L. kirschneri. Additionally, an exact match was observed between serological and molecular data for the few investigated cases where MAT data were available. This methodology is a suitable alternative to the MAT for determining the infecting serovar when Leptospira DNA from blood or urine is detected at Ct values below 34, contributing to clinical surveillance of leptospirosis.

Characteristics of the Urinary Microbiome From Patients With Gout: A Prospective Study.

The role of host microbes in the pathogenesis of several diseases has been established, and altered microbiomes have been related to diseases. However, the variability of the urinary microbiome in individuals with gout has not been evaluated to date. Therefore, we conducted the present prospective study to characterize the urinary microbiome and its potential relation to gout. Urine samples from 30 patients with gout and 30 healthy controls were analyzed by Illumina MiSeq sequencing of the 16S rRNA hypervariable regions, and the microbiomes were compared according to alpha-diversity indices, complexity (beta diversity) with principal component analysis, and composition with linear discriminant analysis effect size. The most significantly different taxa at the phylum and genus levels were identified, and their potential as biomarkers for discriminating gout patients was assessed based on receiver operating characteristic (ROC) curve analysis. Compared with the healthy controls, there was a dramatic decrease in microbial richness and diversity in the urine of gout patients. The phylum Firmicutes and its derivatives (Lactobacillus_iners, Family_XI, and Finegoldia), the phylum Actinobacteria and its derivatives (unidentified_Actinobacteria, Corynebacteriales, Corynebacteriale, Corynebacterium_1, and Corynebacterium_tuberculostearicum), and the genera Prevotella and Corynebacterium_1 were significantly enriched in the urine of gout patients. ROC analysis indicated that the top five altered microbial genera could be reliable markers for distinguishing gout patients from healthy individuals. These findings demonstrate that there are specific alterations in the microbial diversity of gout patients. Thus, further studies on the causal relationship between gout and the urinary microbiome will offer new prospects for diagnosing, preventing, and treating gout.

The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

BACKGROUND: In the past urine was considered sterile. Through the introduction of next generation sequencing, it has become clear that a urinary microbiome exists. Acute kidney injury (AKI) represents a major threat to kidney transplant recipients. Remarkable changes in the urinary metabolome occur during AKI, which may influence the urinary microbiome. To our knowledge, this is the first study that examines the urinary microbiome in renal transplant recipients (RTX) and non-transplant recipients (nRTX) at time of AKI. METHODS: In this cross-sectional pilot-study the urinary microbiome of 21 RTX and 9 nRTX with AKI was examined. Clean catch morning urine samples were obtained from all patients on the first day of AKI diagnosis. AKI was defined according to KDIGO guidelines. Urinary microbiota and the urinary metabolome during AKI were assessed in one patient. 16S rRNA sequencing was performed. Sequences were processed using UPARSE-pipeline for operational taxonomic units (OTU) and taxon finding. RESULTS: We successfully extracted and sequenced bacterial DNA from 100% of the urine samples. All 30 patients revealed at least 106,138 reads. 319 OTU and 211 different genera were identified. The microbiotic diversity richness in the RTX group was no different from the nRTX group. Eighteen genera were solely present in nRTX and 7 in RTX. CONCLUSIONS: The urinary microbiome at time of AKI showed different bacterial genera in RTX compared to nRTX. The nRTX group exhibited no different diversity to the RTX group. Irrespective of the status of a previous renal transplantation, the urinary microbiome comprised > 210 different genera. An intraindividual change in microbiota diversity and richness was observed in one study patient during recovery from AKI.

Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation.

Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device's steel wool matrix for subsequent processing steps. We demonstrate the method's utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5-200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10-17 and 70-96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of ∼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.

Metagenomic analysis of mycobacterial transrenal DNA in patients with HIV and tuberculosis coinfection.

The existence of "transrenal" DNA (tr-DNA), i.e. cell-free DNA that has distributed through the renal barrier to the urine, was first shown from a pathogen in 2000 (Botezatu et al., 2000). However, a targeted search for tr-DNA from Mycobacterium tuberculosis (MBT) started relatively recently (Cannas et al., 2008; Green et al., 2009). While other MBT cellular components found in the urine, e.g. lipoarabinomannan, have been used as an enhanced diagnostic tool, tr-DNA has the potential for strain specific identification or a more persistent biomarker during treatment of active disease. We therefore sought to identify by high-throughput next generation sequencing (NGS) MBT genome fragments in the urine of people with human immunodeficiency virus and tuberculosis (HIV-TB) co-infection living in a co-epidemic setting, and to evaluate whether these DNA targets are suitable for the development a quantitative TaqMan polymerase chain reaction with real-time detection (rt-PCR). Selection and mapping to the reference MBT genome of strain H37Rv (NC_000962) revealed 158 fragments of mycobacterial DNA with length from 19 to 44 base pairs (bp) repeated in different DNA samples. Five targets were chosen for design of rt-PCR primers and probes. Comparative analysis of the newly developed tests that were based on the results of NGS did not reveal a significant increase in sensitivity and specificity relative to the previous empirically designed targets. Howver, highly reproducible NGS reads of mycobacterial tr-DNA were obtained. rt-PCR test development suitable for more practical clinical use was likely limited by the small size of the secreted DNA fragments. It is necessary to develop further molecular approaches for the detection of mycobacterial tr-DNA or rely on NGS techniques with inherent bioinformatics requirements.

Optimizing bacterial DNA extraction in urine.

Urine is an acceptable, non-invasive sample for investigating the human urogenital microbiota and for the diagnosis of sexually transmitted infections. However, low quantities of bacterial DNA and PCR inhibitors in urine may prevent efficient PCR amplification for molecular detection of bacteria. Furthermore, cold temperatures used to preserve DNA and bacteria in urine can promote precipitation of crystals that interfere with DNA extraction. Saline, Dulbecco's Phosphate Buffered Saline, or Tris-EDTA buffer were added to urine from adult men to determine if crystal precipitation could be reversed without heating samples beyond ambient temperature. Total bacterial DNA concentrations and PCR inhibition were measured using quantitative PCR assays to compare DNA yields with and without buffer addition. Dissolution of crystals with Tris-EDTA prior to urine centrifugation was most effective in increasing bacterial DNA recovery and reducing PCR inhibition. DNA recovery using Tris-EDTA was further tested by spiking urine with DNA from bacterial isolates and median concentrations of Lactobacillus jensenii and Escherichia coli 16S rRNA gene copies were found to be higher in urine processed with Tris-EDTA. Maximizing bacterial DNA yield from urine may facilitate more accurate assessment of bacterial populations and increase detection of specific bacteria in the genital tract.

Investigation of Preanalytical Variables Impacting Pathogen Cell-Free DNA in Blood and Urine.

Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. The PCR cycle threshold (CT ) was used to measure amplifiable cfDNA. In spiked samples, the median CT values for M. tuberculosis, S. enterica, and EBV cfDNA were significantly lower in blood collected in K2EDTA tubes than those in Streck and PAXgene blood collection tubes, and they were was significantly lower in urine preserved with EDTA (EDTA-urine) than in urine preserved with Streck reagent (Streck-urine). Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosisCT values than with the Streck blood collection tube and Streck urine preservative. Processing delay increased the median pathogen CT values for Streck and PAXgene but not K2EDTA blood samples and for urine preserved with Streck reagent but not EDTA. Double-spin compared with single-spin plasma separation increased the median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant and between fresh and thawed plasma and urine after 24 weeks at -80°C. Larger plasma and urine volumes in contrived and patient samples showed a significantly lower median M. tuberculosisCT These findings suggest that large-volume single-spin K2EDTA-plasma and EDTA-whole urine with up to a 24-h processing delay may optimize pcfDNA detection.

A cell-free DNA metagenomic sequencing assay that integrates the host injury response to infection.

High-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing, however, does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA (cfDNA) after bisulfite conversion to map the tissue and cell types of origin of host-derived cfDNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cfDNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infection.

Toward the Development of a Circulating Free DNA-Based In Vitro Diagnostic Test for Infectious Diseases: a Review of Evidence for Tuberculosis.

The detection of circulating free DNA (cfDNA) has transformed the field of oncology and prenatal diagnostics. Clinical application of cfDNA for disease diagnosis and monitoring, however, is relatively recent in the field of infectious disease. The potential of cfDNA as a noninvasive diagnostic and monitoring tool is especially promising for tuberculosis (TB), as it enables the detection of both pulmonary and extrapulmonary TB from easily accessible urine and/or blood samples from any age group. However, despite the potential of cfDNA detection to identify TB, very few studies are described in the literature to date. A comprehensive search of the literature identified 15 studies that report detecting Mycobacterium tuberculosis DNA in the blood and urine of TB patients with nongenitourinary disease, but in only six of them were the methodological steps considered suitable for cfDNA isolation and detection. The sensitivities and specificities for the diagnosis of pulmonary and extrapulmonary TB cases reported in these six studies are highly variable, falling in the range of 29% to 79% and 67% to 100%, respectively. While most studies could not meet the performance requirements of the high-priority target product profiles (TPP) published by the World Health Organization (WHO), the study results nonetheless show promise for a point-of-care detection assay. Better designed prospective studies, using appropriate samples, will be required to validate cfDNA as a TB biomarker.

Real-Time, Digital LAMP with Commercial Microfluidic Chips Reveals the Interplay of Efficiency, Speed, and Background Amplification as a Function of Reaction Temperature and Time.

Real-time, isothermal, digital nucleic acid amplification is emerging as an attractive approach for a multitude of applications including diagnostics, mechanistic studies, and assay optimization. Unfortunately, there is no commercially available and affordable real-time, digital instrument validated for isothermal amplification; thus, most researchers have not been able to apply digital, real-time approaches to isothermal amplification. Here, we generate an approach to real-time digital loop-mediated isothermal amplification (LAMP) using commercially available microfluidic chips and reagents and open-source components. We demonstrate this approach by testing variables that influence LAMP reaction speed and the probability of detection. By analyzing the interplay of amplification efficiency, background, and speed of amplification, this real-time digital method enabled us to test enzymatic performance over a range of temperatures, generating high-precision kinetic and end-point measurements. We were able to identify the unique optimal temperature for two polymerase enzymes while accounting for amplification efficiency, nonspecific background, and time to threshold. We validated this digital LAMP assay and pipeline by performing a phenotypic antibiotic susceptibility test on 17 archived clinical urine samples from patients diagnosed with urinary tract infections. We provide all the necessary workflows to perform digital LAMP using standard laboratory equipment and commercially available materials. This real-time digital approach will be useful to others in the future to understand the fundamentals of isothermal chemistries, including which components determine amplification fate, reaction speed, and enzymatic performance. Researchers can also adapt this pipeline, which uses only standard equipment and commercial components, to quickly study and optimize assays using precise, real-time digital quantification, accelerating development of critically needed diagnostics.