Ruchika Mohan

A Biosensor Platform for Rapid Antimicrobial Susceptibility Testing Directly From Clinical Samples

PURPOSE A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test. MATERIALS AND METHODS For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis. RESULTS A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis. CONCLUSIONS This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours.

Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes

Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis

Background Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. Methodology/Principal Findings The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both). Conclusion/Significance We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.

An AC electrokinetics facilitated biosensor cassette for rapid pathogen identification

To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.

Development of a Biosensor-Based Rapid Urine Test for Detection of Urogenital Schistosomiasis.

Schistosomiasis affects up to 300 million people in regions of South America, Southeast Asia, the Middle East, Mediterranean Europe, and sub-Saharan Africa. Chronic consequences of schistosomiasis include anemia, physical and cognitive retardation in children, and organ failure. With predilection for the genitourinary tract, Schistosoma haematobium increases the risk of bladder cancer and HIV infection in women. Microscopic identification and enumeration of parasite eggs in urine (S. haematobium) or stool (Schistosoma mansoni and Schistosoma japonicum) has remained the standard for schistosomiasis diagnosis [1]. Limitations of microscopy include the need for skilled personnel in the field with microscopy equipment and its time-consuming nature. Particularly in infrastructure-limited regions, point-of-care (POC) molecular diagnostics hold the potential to transform the management of infectious diseases such as schistosomiasis that carry significant long-term morbidity if left undiagnosed. POC diagnosis could allow selective drug administration to individuals with confirmed infection rather than entire schools or communities, and an integrated diagnosis–single dose therapy approach could reduce costs of drug administration campaigns and minimize treatment-associated adverse effects. Electrochemical biosensors are well suited for molecular diagnostics because of their high sensitivity, low cost, ease of integration into POC devices, and portability of the reader instrumentation [2]. We have developed a strategy for rapid (one hour) molecular diagnosis of bacterial urinary tract infections using electrochemical biosensors [3,4]. Urinary cells are lysed and directly applied to an array of sensors functionalized with oligonucleotide probes targeting the 16S ribosomal RNA (rRNA) of common uropathogens [3,4]. Formation of the sequence-specific hybridization complex between the pathogen rRNA and the labeled capture and detector probe pairs is detected by an enzyme tag that mediates an amperometric signal output (Fig 1). Fig 1 Biosensor-based molecular detection of urinary pathogen. In this work, we demonstrate the use of our biosensor-based platform for detection of S. haematobium in urine. We developed capture and detector probes targeting S. haematobium rRNA and integrated them into our established molecular diagnostics strategy. After determining an efficient egg lysis strategy, we demonstrated direct electrochemical detection of S. haematobium eggs spiked in human urine.

Integrated Biosensor Assay for Rapid Uropathogen Identification and Phenotypic Antimicrobial Susceptibility Testing

BACKGROUND Standard diagnosis of urinary tract infection (UTI) via urine culture for pathogen identification (ID) and antimicrobial susceptibility testing (AST) takes 2-3 d. This delay results in empiric treatment and contributes to the misuse of antibiotics and the rise of resistant pathogens. A rapid diagnostic test for UTI may improve patient care and antibiotic stewardship. OBJECTIVE To develop and validate an integrated biosensor assay for UTI diagnosis, including pathogen ID and AST, with determination of the minimum inhibitory concentration (MIC) for ciprofloxacin. DESIGN, SETTING, AND PARTICIPANTS Urine samples positive for Enterobacteriaceae (n=84) or culture-negative (n=23) were obtained from the Stanford Clinical Microbiology Laboratory between November 2013 and September 2014. Each sample was diluted and cultured for 5h with and without ciprofloxacin, followed by quantitative detection of bacterial 16S rRNA using a single electrochemical biosensor array functionalized with a panel of complementary DNA probes. Pathogen ID was determined using universal bacterial, Enterobacteriaceae (EB), and pathogen-specific probes. Phenotypic AST with ciprofloxacin MIC was determined using an EB probe to measure 16S rRNA levels as a function of bacterial growth. MEASUREMENTS Electrochemical signals for pathogen ID at 6 SD over background were considered positive. An MIC signal of 0.4 log units lower than the no-antibiotic control indicated sensitivity. Results were compared to clinical microbiology reports. RESULTS AND LIMITATIONS For pathogen ID, the assay had 98.5% sensitivity, 96.6% specificity, 93.0% positive predictive value, and 99.3% negative predictive value. For ciprofloxacin MIC the categorical and essential agreement was 97.6%. Further automation, testing of additional pathogens and antibiotics, and a full prospective study will be necessary for translation to clinical use. CONCLUSIONS The integrated biosensor platform achieved microbiological results including MIC comparable to standard culture in a significantly shorter assay time. Further assay automation will allow clinical translation for rapid molecular diagnosis of UTI. PATIENT SUMMARY We have developed and validated a biosensor test for rapid diagnosis of urinary tract infections. Clinical translation of this device has the potential to significantly expedite and improve treatment of urinary tract infections.

Abstract LB-84: Multiplex detection of urinary biomarkers for rapid bladder cancer diagnosis using an automated cartridge-based platform.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction and Objectives Bladder cancer, a common cancer with high recurrence rate, currently depends on invasive endoscopy for diagnosis and surveillance. A non-invasive, rapid, and accurate urine test will significantly improve management. We have developed a near-patient, fully automated bladder cancer test using the GeneXpert® platform (GX, Cepheid, Sunnyvale CA) for multiplex detection of urinary biomarkers. This test, requires simply mixing voided urine with a liquid preservative followed by loading into the GX cartridge. Materials and Methods Thirty candidate mRNA markers from published studies were selected for evaluation. These markers were screened in urine using RT-qPCR targeting intron spanning amplicons for each candidate mRNA. Urinary expression was measured relative to ABL1 reference gene. Four markers, IGF2, KRT20, ANXA10 and CRH, were chosen based on sensitivity and specificity with analysis of >200 patient cases and control urines. A urine preservative was developed to preserve urothelial cells and their RNA and to lyse red blood cells and white blood cells. Four mL preserved urine was added to an integrated cartridge and inserted into a GX module where the on-board sample preparation steps and RT-qPCR took place. The test results, cancer detected or not, are returned within 90 minutes. With local IRB approval, clinical urine samples were collected from research subjects prior to undergoing cystoscopy for cancer screening or resection. The Xpert® Bladder assay results were compared to urine cytology, tissue histopathology and in selected samples, UroVysion, a commercially available bladder cancer test based on fluorescent in-situ hybridization. Results In our pilot validation study, the Xpert® Bladder assay demonstrated 89.7% sensitivity (61/68) for the detection of high grade bladder cancer; 54.1% sensitivity (33/61) for the detection of low grade bladder cancer; 77.5% specificity (248/320) for cystoscopy negative patients; and 92.7% specificity (51/55) for the healthy control group. In the split sample study the Xpert® Bladder assay demonstrated equal performance compared to UroVysion for patients with high grade bladder cancer and performed better than UroVysion for patients with low grade bladder cancer. Analysis of tumor biopsy tissue from patients whose urine samples were falsely negative showed that all tumors tested had up-regulation of at least one of the four markers, suggesting the failure to detect these markers in the urine may be due to insufficient quantity of urinary tumor cells. Conclusion We have developed a rapid, on-demand urine test for bladder cancer diagnosis based on multiplex detection of a 4-marker panel using a well-established molecular diagnostic platform. Pending further validation, the Xpert® Bladder assay may be integrated as an important part of bladder cancer management. Citation Format: Ellen Wallace, Edwin W. Lai, Kathleen E. Mach, Neda Haque, Leena McCann, Shelly Hsiao, Daniel Bui, Ruchika Mohan, Malini Satya, Edith Wong, Julia A. Bridge, David Persing, Russell Higuchi, Joseph C. Liao. Multiplex detection of urinary biomarkers for rapid bladder cancer diagnosis using an automated cartridge-based platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-84. doi:10.1158/1538-7445.AM2013-LB-84