Broth cultures

A broth culture more accurately represents the growing conditions of bacteria and or fungi. In the broth process the urine is placed into a Trypticase Soy Broth (TSB) for 3-6 days instead of being placed directly onto an agar plate or petri dish for incubation.

Critically, the temperature for the broth mixture is closely controlled which allows the microbes in the urine to evolve as they would in the body.

How does the broth process work?

Urine contains not only bacteria and/or fungi but also body waste products from foods, indigestibles, medications (antibiotics/vitamins) and toxins which need to be cleaned first before testing.

The urine is cleaned, spun for around 20 minutes, so that the residue can be poured away.

The remaining ‘clean’ urine is placed into the broth and incubated at 35 degrees celsius until growth appears. If no growth is found in three days the sample is left for up to another three days.

After the broth process the bacteria are placed onto agar plates and incubated again at 35 degrees Celsius for about 24 hours or until significant bacteria and/or fungal anaerobes emerge on the plate.

Once the infecting bacteria are identified, an ABST (Antibiotic Sensitivity Test) on them is carried out.

The results are provided to the patient so that appropriate treatment can take place.

Broth testing can also provide treatment sensitivities for fungal infections such as candida as well as anaerobes such as lactobacillus.

Please note that United Medical Labs discontinued their broth culture test in May 2019.  This test is now only offered by a couple of UK Chronic UTI specialists.

For more information please see Where to get a UTI Test

A urine dipstick analysis

After you have been asked to provide a mid-stream urine sample, a dipstick is applied to the urine to check for signs of infection or inflammation. This dipstick test strip is made of paper and can have up to 10 different chemical pads or reagents which react (change colour) when immersed in, and then removed from, a urine sample usually after around 60 seconds. The dipstick analysis includes testing for the presence of:

  • White blood cells (known as Leukocytes) – only a few white blood cells are normally present in urine. When these numbers increase, the dip test will become positive. This indicates that there is inflammation in the urinary tract or kidneys and the body is excreting more white blood cells. White blood cells also produce antitoxins that neutralise the toxins released by bacteria.
  • Red blood cells – the bladder can bleed due to severe inflammation and the constant urination caused by a UTI. Some people can feel a “razor blade sensation” when urinating during a UTI attack.
  • Protein – the presence of protein can indicate a possible kidney infection as only trace amounts normally filter through the kidneys. Other causes of protein in the urine can include kidney disease, dehydration,
  • Nitrites– Nitrite tests detect the products of nitrate reductase, an enzyme produced by some bacterial species that can cause a UTI. These products are not present normally unless a UTI exists. A positive result on the nitrite test is highly specific for UTI, typically because of urease-splitting organisms, such as Proteus species and, occasionally, E coli; however, it is very insensitive as a screening tool, as only 25% of patients with UTI have a positive nitrite test result. This occurs in cases with low bacterial colony forming unit counts, or in recently voided or dilute urine as it can take up to four hours before the urinary enzyme nitrate reductase is converted by bacteria in urine.  In addition, a nitrite test does not detect organisms unable to reduce nitrate to nitrite, such as enterococci or staphylococci.

Any evidence of these in the urine on a dipstick test indicates bladder inflammation/infection.

Additionally, the dipstick reagent pads test for the presence of Bilirubin, Urobilinogen, Ketones and Glucose.

Find out more about how to interpret your urine dipstick test.

Expanded Quantitative Urine Culture (EQUC)

Increasingly used in research studies investigating the urinary microbiome and how it influences recurrent and chronic UTI, Expanded Quantitative Urine Culture (EQUC) is a new technology using a used a modified culture protocol from that of the standard urine analysis laboratory method.  This includes analysing larger volumes of urine (10–100 mL instead of 1 mL for a standard mid stream urine culture), the incubation of samples using differing atmospheric conditions to cultivate bacteria that may not grow on standard urine culture and longer times for incubation (up to 48 hours).

A study by Loyola University Chicago, published in the American Journal of Microbiology in 2014 comparing the standard urine culture against EQUC noted that the EQUC analysis method found bacteria in 92% of samples tested compared against the same samples analysed via standard methods which showed negative growth for bacteria. With this, bacterial presence as low as 10 colony forming units (CFU) per mL could be detected; indeed, Lactobacillus, Corynebacterium, and multiple other genera were isolated using this EQUC. The authors proposed a “streamlined” version of this EQUC, which specified using a higher volume of 100 mL of urine on MacConkey, blood, and colisitin-nalidixic acid (CNA) agars in a 5% CO2 incubator for 48 h to yield 84% sensitivity relative to the extended spectrum protocol. This protocol has been utilized by several investigators since.

A further study of 150 women published in the Journal of Clinical Microbiology in 2016, patients were grouped by whether or not they had self-reported UTI-like symptoms and performed the aforementioned streamlined EQUC on catheterized urine specimens; while they did not find a difference in the number of isolated uropathogens, they did find a reduced species richness and diversity in patients who did have clinical UTI symptoms. About half of the uropathogens in the UTI cohort were missed by standard urine culture; additionally, the threshold of 105 CFU/mL would not report a predominant organism in numerous patients with a clinical UTI in this cohort.

Mid-Stream Urine Culture (MSU)

The current laboratory test to investigate for a bacterial UTI involves providing a fresh urine sample. This is known as a mid-stream urine culture or MSU. To get an accurate result and avoid bacterial contamination, a “clean” midstream urine sample is used. This means you don’t collect the first or last part of the urine stream but only the middle part by interrupting the flow of urine after a few seconds and then collecting this middle part of the urine flow in the sample bottle provided. Once collected, the sample is sent by your GP or consultant to the laboratory for analysis.

How your urine is analysed in the laboratory

What are the issues with a standard mid-stream laboratory culture?

Fresh urine microscopy

A sample of fresh urine is dropped onto a plate for immediate analysis under a microscope. White blood cells and epithelial cells are counted as markers of an infection. When infection occurs in the urinary tract, the immune system tries to remove infected cells by shedding the cells in the bladder lining (the epithelium) to be excreted during urination – these are known as epithelial cells.

A high white blood cell count usually indicates that the body is fighting an infection. White blood cells rush in to help destroy the harmful substance and prevent the infection developing further.

White blood cells degrade very quickly. Studies have shown this occurs in as little as four hours unless stored in the correct conditions so a sample sent for analysis at the laboratory will not pick them up.

Professor James Malone-Lee, Emeritus Professor of Nephrology, UCL notes in two published studies: “Analysis of urothelial and clue cells is novel, as is sediment culture, but these methods have been well validated and have a strong pathophysiological foundation. Fresh urine microscopy is not commonly adopted in clinical practice nowadays, nevertheless, it has been well validated in studies dating back to 1928 and is still unsurpassed as a surrogate marker of infection”. (1)

“Pyuria, detected by microscopy of a fresh midstream urine (MSU) specimen, is the most sensitive surrogate marker of UTI. It circumvents the problems associated with quantitative (numerical) bacterial culture, and its value in the diagnosis of UTI is recognised by international practice guidelines. Whilst ≥10 wbc in a micro litre of urine is employed almost universally to diagnose UTI, contemporary data cast doubt on this threshold in patients with LUTS. In the symptomatic patient, controlled studies have demonstrated that lower pyuria counts of 1–9 wbc in a micro litre of urine are associated with an increase in independent inflammatory and microbiological markers of UTI. Thus, lower levels of pyuria may also indicate infection and immune activation”.(2)

Issues with urine microscopy

  • Urine samples must be fresh, that is within two hours of urination to prevent degradation of the white blood cells otherwise the sample must be discarded
  • Training of staff to interpret samples via microscopy plus the cost of the relevant microscope
  • Acceptance by clinicians of the results and then appropriate treatment prescription. At present urine testing is biased towards bacterial identification. Urine microscopy, once the standard for urine analysis, was superseded by the automated methods introduced since the 1960s preferred for their time-saving benefits.

References:

1. A blinded observational cohort study of the microbiological ecology associated with pyuria and overactive bladder symptoms. Kiren Gill, Ryoon Kang, Sanchutha Sathiananthamoorthy, Rajvinder Khasriya, James Malone-Lee. International Urogynecology Journal, January 2018

2. Recalcitrant chronic bladder pain and recurrent cystitis but negative urinalysis: What should we do?  Sheela Swamy, William Barcella, Maria De Iorio, Kiren Gill, Rajvinder Khasriya, Anthony S. Kupelian,  Jennifer L. Rohn, James Malone-Lee. International Urogynecology Journal, March 2018

Next Generation Sequencing (NGS)

Bacteria can be classified using conventional microbiology methods, such as microscopy or being grown on specific media in a laboratory and using antibiotic sensitivity assessments. In recent decades, molecular microbiology methods have revolutionized bacterial identification. A popular method is 16S ribosomal RNA (rRNA) gene sequencing. This method is not only faster and more accurate than conventional methods, but also allows identification of strains that are difficult to grow in standard laboratory conditions.

The 16S rRNA gene is present in all bacteria. This makes it an ideal genetic fragment to be used in identification and comparison of bacteria. NGS is a process which uses this 16S rRNA gene identification to analyse for quality. Only the highest quality bacteria, anaerobes or fungi identified will be interpreted and reported. The data for each detected bacterial or fungal species is then reported as a percentage of specimens identified.

Deep NGS or Shotgun sequencing goes further, it creates a genetic fingerprint of everything in your urine sample and is able to identify tens of thousands of microorganisms in one sample. This can include not only bacteria but parasites and viruses something that PCR and simple 16S NGS testing cannot offer.

Benefits of NGS testing

  • NGS tests are not affected by temperature or time delays unlike traditional urine cultures where the urine sample must be delivered to the laboratory within two hours to prevent sample degradation. Often standard cultures will return a no growth result when samples have not arrived at the laboratory within the time frame required for successful culturing.
  • NGS testing does not grow microbes, they are instead extracting the microbial DNA from a sample and can identify multiple bacteria, anaerobes or fungi. They require just need one sample.
  • These reports will be comprehensive in nature covering all micro-organisms in your sample rather than focusing on a single pathogen which the standard laboratory culture is directed towards.

Issues with NGS testing

There are some uncertainties about NGS testing in a clinical setting:

  • All testing is reliant on the sample containing the presence of biofilm pieces or infected bladder lining cells.  If these are not present in the sample you submit, no laboratory test offered by these companies will be able to detect the pathogens contained within them. Dormant, embedded bacterial DNA will not be detected.
  • At present, it isn’t possible for a DNA sequencing test to tell you whether any micro-organisms identified were part of a biofilm or intracellular community or not.
  • It is currently unknown how the urinary microbiome may differ from the microbiome of the bladder wall and in particular those bacteria embedded into bladder wall cells or those affected by biofilm coverage. To understand the differences further will likely require more invasive sampling procedures such as biopsies.
  • The DNA extraction technique chosen will significantly affect how faithfully the bacterial composition of the original sample is represented by the DNA extracted from it. In some bacteria, such as Gram-positive bacteria and Mycobacteria, it can be more difficult to break down the cell membrane of the bacteria (known as lysis) for study than others in a microbial community and these bacteria may be less represented in a report. On the other hand, if an extraction method is too harsh, the DNA from the easily lysed species may become sheared. Currently, there is no standard technique that works equally well for lysing all bacteria in a given sample.
  • Another limitation of sequencing-based approaches is that they only yield information about the microbial DNA in a sample. While this allows for identification of the types of bacteria present in a biological sample, it does not distinguish between bacteria that are live from those that are dead.
  • NGS reports give percentages on the bacteria/fungi or microbes found in your urine. But the understanding of the urinary microbiome is still in development. Are all those identified responsible for your urine infection? Does a higher percentage equate to the causative agent behind your infection?
  • The testing methods are based on individual, unique, patented methods used by each laboratory.  They test against a panel of microbes/fungi/anaerobes established by that company.  If the infection causing micro-organism is not found against that panel, how are you to be treated?
  • There is also the issue of cost. Treatment of a chronic UTI may involve several NGS tests over a period of time and at present, these are extremely expensive. Some patients in the US are able to offset their costs through health insurance but others are unable to do so. International patients cannot offset against health insurance.
  • It can be difficult to find a practitioner who can interpret the laboratory findings and treat accordingly. Treatment guidelines used by GPs and consultants worldwide are based on standard testing protocols. Some patients have found their results have been dismissed or ignored by their doctor or consultant.
  • Finally, as yet, no research studies have been published using NGS testing in a clinical setting to determine success outcomes for patients using this diagnostic method.

PCR or polymerase chain reaction testing

PCR testing (polymerase chain reaction) is a technique used to amplify trace amounts of DNA found in or on almost any liquid or surface.

Every human, animal, plant, parasite, bacteria or virus contains genetic material such as DNA sequences that are unique to their species.

If a urine sample contains segments of DNA, PCR is a method used to amplify (make many more identical copies) of these unique sequences to determine with a very high probability the pathogenic bacteria, fungi or other microbes.

Within urine, PCR testing has detected the DNA of more than 1,200 microbial species.

Benefits of PCR testing

  • PCR tests are not affected by temperature or time delays unlike traditional urine cultures where the urine sample must be delivered to the laboratory within two hours to prevent sample degradation.
  • The results of a PCR test can be available within 24 hours.
  • PCR tests do not grow microbes, they are instead extracting the microbial DNA from a sample and can identify multiple bacteria, anaerobes or fungi.
  • A PRC test needs one sample unlike standard laboratory tests where a separate swab would be required for fungal microbe analysis in addition to a urine sample.  This fungal analysis can take up to 20 days to process.

Limitations of PCR testing

  • Laboratories using this method need to preselect which microorganisms the test looks for.  Urine panels usually comprise of less than a dozen micro-organisms, more often the most common for that type of bacterial infection. Any other organisms not listed in this panel will be excluded from analysis.  It amounts to around 1% of all known microbes.
  • It is currently unknown how the urinary microbiome may differ from the microbiome of the bladder wall and in particular those bacteria embedded into bladder wall cells or those affected by biofilm coverage. To understand the differences further will likely require more invasive sampling procedures such as biopsies.
  • The DNA extraction technique chosen will significantly affect how faithfully the bacterial composition of the original sample is represented by the DNA extracted from it. In some bacteria, such as Gram-positive bacteria and Mycobacteria, it can be more difficult to break down the cell membrane of the bacteria (known as lysis) for study than others in a microbial community and these bacteria may be less represented in a report. On the other hand, if an extraction method is too harsh, the DNA from the easily lysed species may become sheared. Currently, there is no standard technique that works equally well for lysing all bacteria in a given sample.
  • Another limitation of sequencing-based approaches is that they only yield information about the microbial DNA in a sample. While this allows for identification of the types of bacteria present in a biological sample, it does not distinguish between bacteria that are live from those that are dead.
  • In comparison to Next Generation Sequencing testing it is thus limited because it won’t offer a genetic report of everything in the urine sample you provide.
  • PCR reports give percentages on the bacteria or microbes found in your urine. But the understanding of the urinary microbiome is still in development. Are all those identified responsible for your urine infection? Does a higher percentage equate to the causative agent behind your infection?
  • The testing methods are based on individual, unique, patented methods used by each laboratory. They test against a panel of microbes/fungi/anaerobes established by that company. If the infection causing agent is not found against that panel, how are you to be treated
  • There is also the issue of cost. Treatment of a chronic UTI may involve several tests over a period of time and at present, these are extremely expensive. Some patients in the US are able to offset their costs through health insurance but others are unable to do so. International patients cannot offset against health insurance.
  • It can be difficult to find a practitioner who can interpret the laboratory findings and treat accordingly. Treatment guidelines used by GPs and consultants worldwide are based on standard testing protocols. Some patients have found their results have been dismissed or ignored by their doctor or consultant.
  • Finally, as yet, no research studies have been published using PCR testing in a clinical setting to determine successful treatment outcomes for patients using this diagnostic method.