Thanks to their readily non-invasive availability, urine samples are among the oldest known diagnostic specimen types. Egyptian papyri from ~1350 BC describe a urine-based test for pregnancy and fetal gender. (Remarkably, modern evaluation of the assay described showed it to be about 70 percent accurate at evaluating early pregnancy status, although the claim of gender determination didn’t hold up.) Modern clinical practice includes chemical urinalysis for a number of applications. But what about urine’s potential as a specimen for MDx applications? Do diagnostically meaningful DNA or RNA molecules occur in urine, and if so, are there any special sample handling considerations for molecular analysis of urine? Spoiler alert: the answers will be “yes” and “yes,” but let’s examine both questions in more detail.
Cellular material in urine
Perhaps the most obvious source for nucleic acids in a urine sample is within any cells present. Both red and white cells are normally observed at low levels in urine. The former is of little interest to us as they are missing nuclei, but the latter contains a full complement of DNA for potential analysis. Given the usual ready availability of other, higher cellular content genomic DNA samples types, such as buccal swabs or residual peripheral blood samples already taken for other tests, though, this doesn’t seem very useful as an MDx specimen.
A more interesting second source of cells in urine is the sloughed cells of the bladder or urinary tract. In cases of suspected bladder cancer, analysis of these cells for particular genetic aberrations common to bladder cancers (including aneuploidy of chromosomes 3, 7, and/or 17, or loss of the 9p21 region) can be a useful tool in assisting diagnosis or monitoring for recurrence following treatment. FISH (fluorescent in-situ hybridization) is the MDx method basis for at least one such FDA-approved test. FISH allows for direct visualization of the absence or presence and number of particular chromosomal regions through the specific hybridization of fluorescently labelled probes and examination on common laboratory fluorescence microscopes. (Readers unfamiliar with this technique who wish to read a brief discussion are directed to the August 2015 “Primer” article, “Gone FISHing: a cytogenetic interlude“). Methylation status at a handful of genome loci has also been proposed as a diagnostic tool for bladder cancer, and an assay under commercial development which evaluates this has published promising preliminary results.
A third potential source of nucleic acids in urine is bacterial or fungal cells, as found in the case of urinary tract infections (UTIs). While these are most frequently assayed for and analyzed by classical methods (urine culture), MDx approaches to the identification and enumeration of urine sample pathogens do exist. Most are based on PCR detection of particular pathogen-specific sequences and offer significant benefits over culture both in turnaround time (a few hours versus up to 48 hours) and the potential to detect and enumerate specific non-culturing organisms of interest.
As UTIs are a classic example of multiple “common suspect” pathogens possibly having similar presentations, this application naturally lends itself to multiplex test formats that can simultaneously interrogate the sample for an entire list of pathogens.
Recent publications of clinical evaluations of laboratory developed tests (LDTs or “homebrew” assays) appear promising, such as that by van der Zee and colleagues, in which a semi-quantitative assay capable of detecting and enumerating E. coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., P. mirabilis, E. faecalis, and P. aeruginosa was shown to be appreciably more sensitive than culture.1 Note that, as in this example, the use of a quantitative (or semi-quantitative) molecular method is much preferred over a purely qualitative assay in the context of UTI diagnosis, as low levels of bacterial or fungal DNA can be present without being clinically relevant. (For comparison, the reference culture method requires 10^3 cfu/ml to diagnose cystitis and 10^5 cfu/ml for pyelonephritis, according to guidelines from the Infectious Disease Society of America.2) Another organism for which molecular testing on urine samples shows particular promise is C. trachomatis.3,4
Pathogens detectable via urine are not solely restricted to bacteria and fungi. Parasites represented by Schistosoma species have been successfully reported as detected this way, and several studies have reported detection of HPV (and particularly E6 and E7 RNA expression) from this matrix as a sensitive yet readily obtainable alternative to genital tract swabs. Zika virus, an emerging pathogen that is subject to much current interest, appears to be more reliably detected by RT-PCR on urine than on serum samples; a very recent study by St. George and associates showed that out of 80 positive cases, 66 were detected in urine samples, while only 30 were detected in serum.5 (This may, however, be influenced by the stage of infection; for comment in this context the reader is directed to reference 6).
Lest we start to think that all manner of pathogens can be effectively detected in urine, however, it’s worthwhile to temper this list with at least one noteworthy counterexample, perhaps best provided by B. burgdorferi. Despite some early reports of utility in this context, urine MDx appears to be particularly unreliable and insensitive for this application. As always, no one specimen does everything, and good communication between the lab and collecting physicians is important in matching sample type to target to be detected.
Nucleic acids in urine
We’ve established that cellular material in urine can be an MDx specimen of interest in at least some situations; but are there acellular nucleic acids present in urine, and are they of any potential diagnostic interest? A first application of this is in the context of renal transplant, where elevation of donor organ-derived cell-free DNA in urine is reported as an effective marker of acute transplant rejection. (It is most easily performed in the context of transplant of male-derived kidneys into a female graft recipient; detection of Y-chromosome sequences allows for a ready marker of organ-derived DNA as distinguishable from host DNA without resorting to sequence analysis.) Use of these same holandric (Y-linked) markers has also demonstrated that cell-free DNA derived from peripheral blood circulation is also routinely present and examinable in urine samples; for example, following blood transfusion, donor-derived DNA is detectable in urine; similarly, the detection of single-copy fetal genes has been reported in urine of pregnant mothers.
Encouraged by these results, researchers have successfully demonstrated detection of various tumor-specific DNAs (from non-urogenital tract tumors, as distinct from the more obvious cases such as bladder cancer discussed above). Whether urine is a good surrogate for direct peripheral blood sampling in the search for what are expected to be blood-borne nucleic acids is another question; certainly, target concentrations would be expected to be much higher (and risk of inhibitors lower) in blood samples than in urine samples. In most such cases, improved sensitivity would seem to outweigh the minimal risks associated with blood sample collection, and “feasible” doesn’t necessarily mean “good.” In low-resource settings where risks associated with blood sample collection increase, however, the calculus may shift more in favor of urine specimens.
Sample collection and preparation
The ease of collection offered by urine sampling is offset by the frequent occurrence of MDx inhibitory substances in these samples, with PCR inhibition being observed in the range of five percent to 12 percent of samples. (For unknown reasons, urine from pregnant female subjects has significantly higher inhibition rates than from non-pregnant controls.) Foremost by prevalence among known inhibitors in this specimen type is urea, almost universally present at concentrations where significant polymerase inhibition occurs (depending both on sample factors such as urine volume and diet, and on reaction factors such as buffer composition and specific polymerase being used).
While other inhibitory substances are almost certainly present as well, urea alone is enough to make sample preparation critical to the sensitivity of using urine as an MDx specimen. Some published protocols suggest that overnight sample freezing followed by 1/10 dilution is sufficient to allow for direct PCR, while other, more recent, studies have suggested that freezing and subsequent thawing of urine samples can markedly reduce sensitivity of some MDx assays compared to fresh, unfrozen samples.7 Given that DNases are found endogenously in urine, a plausible hypothesis in this case (a schistosomycete) is that a freeze/thaw cycle may disrupt target organism membranes and allow DNA exposure and subsequent degradation. Regardless of whether this is the underlying true cause, the accepted facts that there are both common MDx inhibitors and nucleases present in urine suggest that samples to be used for any MDx application should ideally be collected fresh and subjected to some form of nucleic acid extraction and purification process as expeditiously as possible to get good assay performance.
One final topic is the obvious potential for contamination of urine samples with exogenous bacteria. Suffice it to say that this is a well characterized issue from the context of traditional urine culture, and the same sampling techniques developed there to minimize this risk (“clean-catch midstream”) should be employed for MDx. Where the testing is done in context of a possible UTI, this further highlights the utility of a (semi) quantitative MDx method over a purely qualitative approach.
REFERENCES
- van der Zee A, Roorda L, Bosman G, Ossewaarde JM. Molecular diagnosis of urinary tract infections by semi-quantitative detection of uropathogens in a routine clinical hospital setting. PLoS ONE. 2016;11(3): e0150755. doi:10.1371/journal.pone.0150755.
- Hooton TM, Bradley SF, Cardenas DD, et al. Diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America. Clinical Infectious Diseases. 2010; 50:625–663. PMID: 20175247.
- Haugland S, Thune T, Fosse B, Wentzel-Larsen T, Hjelmevoll SO, Myrmel H. Comparing urine samples and cervical swabs for chlamydia testing in a female population by means of Strand Displacement Assay (SDA). BMC Women’s Health. 2010;10:9. doi:10.1186/1472-6874-10-9.
- Young H, Moyes A, Horn K, Scott GR, Patrizio C, Sutherland S. PCR testing of genital and urine specimens compared with culture for the diagnosis of chlamydial infection in men and women. Int J STD AIDS. 1998;9(11):661-665.
- St. George K, Sohi IS, Dufort EM, et. al. Zika virus testing considerations: lessons learned from the first eighty real-time RT-PCR-positive cases diagnosed in New York State. J Clin Microbiol. 2016;Dec 7. pii: JCM.01232-16.
- Wiwanitkit, V. Int Urol Nephrol.2016;48:2023. doi: 10.1007/s11255-016-1417-6.
Fernández-Soto P, Velasco Tirado V, Carranza - Rodríguez C, Pérez-Arellano JL, Muro A. Long-term frozen storage of urine samples: a trouble to get PCR results in Schistosoma spp. DNA detection? PLoS ONE. 2013;8(4): e61703. doi:10.1371/journal.pone.0061703.
John Brunstein, PhD, is a member of the MLO Editorial Advisory Board. He serves as President and Chief Science Officer for British Columbia-based PathoID, Inc., which provides consulting for development and validation of molecular assays.
