The Need for Faster UTI Pathogen Detection

Urine culture is the gold standard, but labs can accelerate detection by using molecular methods

Nischal PM, PhD

Nischal PM, PhD, is a science and medical writer. He has a background in medical microbiology and has a keen interest in infectious diseases and antimicrobial drug resistance.

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Published:Mar 26, 2020
|Updated:Jun 02, 2023
|5 min read

Urinary tract infections (UTIs) are one of the most common bacterial infections, both in the community setting and in the hospital environment. At least 50 percent of the population will have an episode of UTI during their lifetime. UTIs cause significant morbidity in females of all age groups, and in young and elderly males. In the US, the collective costs of UTIs, including healthcare expenditure, is approximated at US$3.5 billion per year.

Clinical Lab Manager Pathogen Detection Survey  

The most common causative agents of UTIs are members of the Enterobacteriaceae, including Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Enterobacter species. Fungal infections are more common in patients with indwelling catheters, diabetes, and recent antibiotic usage. Other less common causes include Mycobacterium tuberculosis and parasites such as Schistosoma haematobium. Many of these pathogens are increasingly multidrug-resistant as a result of the misuse of broad-spectrum, empiric antibiotic therapy. 

UTIs, if left untreated, can lead to serious sequelae including pyelonephritis, renal damage in the young, and colitis due to Clostridioides difficile. There is, thus, the need to move from the current time-consuming detection methods towards newer technologies that will advance and accelerate detection.

Presumptive diagnosis of UTI

Most urine samples that are sent to the clinical laboratory are negative for pathogens. Hence, to improve both the laboratory workflow and to reduce costs, clinical laboratories perform initial screening for bacteriuria. Currently available methodologies for the presumptive diagnosis of UTIs include urine dipstick tests, microscopic urinalysis using flow cytometry, and urine Gram-staining.

Urine dipstick tests

Dipstick tests are used to detect nitrites (bacteria convert urinary nitrates to nitrites) and leucocyte esterase (an enzyme produced by neutrophils, which presumptively associates pyuria with an UTI). They are advantageous in being simple and quick to use. 

These tests have several disadvantages, including low sensitivity. False negative nitrite tests are common due to insufficient bladder incubation time for converting nitrate to nitrite, reduced urinary excretion of nitrate, and the failure of certain pathogens, particularly Enterococcus faecalis, to convert nitrate to nitrite. 

Microscopic urinalysis

Flow cytometry can be used for the initial screening of urine samples. Depending on the sample, the concentration of bacteria required for a positive result may range from 40 to 1000/µL.  

The advantages of urine flow cytometry are that it is standardized, less expensive when compared to urine culture, and the results are rapid. The disadvantages are that flow cytometry is only a screen for bacteriuria; also, it does not provide either species identification or antimicrobial susceptibility testing (AST). 

Urine Gram-staining

The analysis of Gram-stained urine samples can be used as a screening technique for UTI. The sensitivity depends on whether the sample has been centrifuged or not. While the urine Gram-stain test provides quick information on the nature of the urinary pathogen, it is disadvantageous in that it is labor intensive and the sensitivity is poor. It is reliable only when the bacterial concentration in urine is >10colony forming units per milliliter (CFU/mL). 

Definitive diagnosis of UTI

The gold standard

The diagnostic gold standard for clinically relevant UTIs continues to be urine culture and identification. However, the disadvantages are that it is laborious, expensive, and time-consuming, with a minimum turnaround time of 24 to 72 hours. This delay is an issue because when the UTI is left untreated for too long, it can lead to serious conditions such as renal damage and colitis. 

Despite the various disadvantages, urine culture continues to be used widely, as it gives an estimate of bacteriuria levels as well as isolated colonies that are used for pathogen identification and antimicrobial susceptibility testing (AST).

Microbial genetics to the fore

Rapid advances in molecular biology have enabled the detection of pathogens based on their molecular signature. Additionally, the genetic basis of antimicrobial drug resistance has been revealed for most clinically relevant pathogens. Hence, the rapid identification of pathogens and their drug resistance patterns has a crucial role to play in the detection of UTIs and other diseases.

The following are microbial genetics methods that have been approved over the last two decades for the diagnosis of bacterial infections and applied to rapid diagnosis of UTIs from urine samples.

MALDI-TOF mass spectrometry

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was initially approved for pathogen identification in UTIs, wherein the urine sample is cultured, the bacteria isolated, and a colony from the culture plate then analyzed. For the direct analysis of urine, several initial sample preparation steps are essential. These include the removal of cellular debris, leukocytes, and mucus, and the collection of bacteria. Best results are obtained when there is a high bacterial count (>105CFU/mL) and Gram-negative bacteria are involved. The main disadvantage is the inability to perform AST, for which conventional methods will still be required.

Fluorescence in situ hybridization (FISH)

The target for bacterial detection in FISH is the 16S ribosomal RNA (rRNA), which is an integral constituent of bacterial genomes and is present in abundant quantities in the bacterial cell. Detection depends on the availability of specific probes.  Rapid FISH assays are available that can give results within 20 minutes with high sensitivity and specificity. The main disadvantage is the inability to perform AST, for which conventional methods will still be required. 

Multiplex PCR

Multiplex PCR assays are increasingly being used in the clinical laboratory for the rapid detection and identification of uropathogens. The advantage is the ability to identify organisms that are not detected by routine culturing because they are considered fastidious. The disadvantage with currently available multiplex PCR assays is that they do not provide comprehensive or definitive phenotypic information about antibiotic susceptibility.

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Emerging diagnostic platforms

The new kids on the block in UTI detection are biosensors, microfluidics, and lab-on-a-chip technology. They have the potential to accelerate the diagnosis of UTIs with enhanced screening, identification of pathogens, and rapid AST.


With advances in microtechnology and nanotechnology, biosensors with integrated microfluidics handling systems have been developed that are capable of doing complex molecular assays necessary for pathogen detection in biological matrices.

Biosensors typically have two components—a recognition element (antibodies or nucleic acids) and a signal transducer. When the target analyte binds to the recognition element, a specific signal is generated, which is then detected by various techniques such as opticalor electrochemical conductance. There are two types of detection strategies—label-free and labeled assays.

The chief advantage of biosensor assays is the rapid turnaround time. Both pathogen identification and AST can be completed within one and 3.5 hours, respectively. The major limitation with currently available biosensor assays is the need for manual sample preparation steps.


Microfluidics is a multidisciplinary field wherein small amounts of fluids are manipulated at the micron scale. Microfluidics-based platforms are a type of lab-on-a-chip platform, which is a miniaturized device that integrates one or more analyses onto a single chip.

Among the benefits of using a microfluidics platform are the ability to integrate complex workflows, compactness of devices and associated instruments, and the low volume of sample and reagents required. The drawbacks of this system are the costs involved and the need for advanced training by laboratory personnel.


Urinary tract infections continue to be a major public health problem in both the community and the hospital setting. The current gold standard for detection of UTIs is urine culture followed by AST. The final results are available only after 24 to 72 hours, which delays the initiation of targeted antimicrobial therapy. 

There is an urgent need for fast and accurate diagnosis of UTIs that will, in turn, lead to timely and effective therapies. In this regard, technologies such as mass spectrometry, FISH, and multiplex PCR, along with newer technologies such as biosensors and microfluidics are a key step toward rapid and improved identification of uropathogens and their antimicrobial sensitivity patterns, and improved antimicrobial stewardship.