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Effect of refrigeration of clinical canine urine samples | VETgirl Veterinary Continuing Education Podcasts

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In today’s VETgirl online veterinary continuing education podcast, we discuss the effect of refrigeration on clinical canine urine samples and quantitative bacterial culture.

Urinary tract infections (UTIs) are a relatively common condition we see in small animal veterinary medicine. UTIs produce classic clinical signs such as polyuria and dysuria. Diagnostics typically include sterile urine collection (e.g., cystocentesis), urine culture, and potentially advanced imaging (e.g., abdominal ultrasound, radiographs, etc.). Urinalysis will unveil the bacterial offenders and culture and sensitivity of the urine sample improves our success in treating this condition. Since most of us do not work in practices with the capability of performing immediate bacterial cultures, we often have to ship our urine samples to the nearest clinical pathology lab. As clinicians, we need to consider how storage and handling of the urine samples will affect the results of our urine cultures. It is common practice in veterinary hospitals to store urine samples collected within the day in glass tubes under refrigerated conditions until they can be batch shipped to the clinical pathology laboratory for culture. So, Acierno et al out of Louisiana State University wanted to evaluate this in a study entitled “Effect of refrigeration of clinical canine urine samples on quantitative bacterial culture.” In this study, the authors aimed to determine if storing urine in glass tubes under refrigerated conditions for 24 hours would affect the yield of colony forming units as compared to culturing the urine immediately. The authors also sought to determine if bacterial yield could be improved under these stored conditions by using a bacterial enrichment broth prior to storage in refrigerated samples.

This was a prospective study that evaluated urine samples collected via cystocentesis from client owned dogs at the Louisiana State University Small Animal Hospital. Dogs in this study had presented to the veterinary hospital with clinical signs consistent with a UTI. Dogs on antimicrobials were excluded from the study. From each canine urine sample, a 1mL aliquot was refrigerated in a glass tube at 5°C to mimic our common storage conditions prior to handling at a clinical pathology laboratory. Half of a milliliter of collected urine was added to 3mL of tryptic soy broth prior to refrigeration at 5°C. Another half of a milliliter of collected urine was immediately cultured. Both refrigerated samples were cultured after 24 hours of refrigeration. Cultures were performed as follows: 5 serial dilutions were made for each sample between 1:10 and 1:100,000 and spread on MacConkey agar and blood-supplemented tryptic soy agar plates. After inoculation, plates were incubated in bench top incubators for 24 hours at 37°C. The blood agar plates were then used for quantification of CFUs unless Proteus spp were recovered as this bacteria is motile and will cover the plate so colonies cannot be counted. Bacteria were identified via standard technique using gram staining and other specialized testing methods.

104 canine urine samples were evaluated in this study. Bacterial growth was identified in 35.6% (37/104) of immediately processed samples. Bacterial growth was identified in 33.7% (35/104) of samples that were first refrigerated in glass tubes without TSB growth media and in 31.7% (33/104) of samples that were inoculated into TSB broth and then refrigerated. This study found that for every urine sample aliquot that had been refrigerated and successfully yielded positive bacterial growth, the concurrent aliquot of urine from the same sample that was immediately cultured out also yielded positive bacterial growth. However, not all urine samples that yielded positive growth with immediate inoculation had concurrent positive growth in the aliqouts that were refrigerated prior to culture. Occasional false negative growths occurred when the aliquots were refrigerated with or without TSB. Refrigerated aliquots produced a high level of sensitivity at 95% (35/37) and 100% specificity. Surprisingly, the act of inoculating TSB broth with the urine sample prior to refrigeration resulted in a lowered sensitivity at 89% (33/37). There were no false positive growths in the refrigerated samples. There was no significance found in CFU yields between immediately inoculated and refrigerated samples, but the tryptic soy broth did produce a significantly lower CFU yield than the immediately inoculated samples. As we would expect, the most commonly isolated bacteria species were E. coli (14) and Staphylococcus spp (13). These were followed in occurrence by Klebsiella spp (7), Enterococcus spp (5), Pseudomonas spp (1), Proteus spp (1), and Streptococcus spp (1). This study found that female urine samples were significantly more likely to have positive bacterial growth than males.

The authors of this study conclude that although refrigeration of urine samples for 24 hours yielded 95% sensitivity and 100% specificity for bacterial growth, it is still best to inoculate urine into culture media immediately for best yields. The authors conclude that tryptic soy broth should not be used to improve yields in refrigerated samples, but VETGirl suggests that further testing with tryptic soy broth may be needed to evaluate whether this step could enhance bacterial yields. Tryptic soy broth is typically incubated after inoculation to match the environmental needs of the bacteria (i.e. incubating at 35°C for E.coli species to mimic body temperatures). In the present study, the tryptic soy broth samples were inoculated and then refrigerated and cultured immediately after 24 hours of refrigeration without incubation time in the broth at optimal temperatures for the bacteria. By taking a small aliquot of urine and dropping it into 3 mL of TSB broth, the bacteria present in the urine was diluted into the broth. Then by refrigerating the samples, the bacterial growth was inhibited.1,2 So it would appear that this extra step understandably resulted in a diluted urine sample (thereby diluted bacteria burden) and produced the lower percentage of bacterial yield as compared to undiluted urine under refrigerated and non refrigerated conditions.

So, what do we take away from this VETgirl podcast?

It’s still ok to do what we are currently doing. Collecting our urine samples and storing in glass tubes under refrigerated conditions until they can be batch shipped that day still produces really good yield and culture results. In an ideal setting, the urine samples would be submitted immediately for culture at the lab or would be inoculated onto growth media immediately within the practice. Using an enrichment broth is not presently recommended for storing specimen samples such as urine samples under refrigerated conditions. Again, this is likely because, in this study, the tryptic soy broth was inoculated and then immediately refrigerated, with no incubation period at warmer temperatures prior to culturing. Essentially, what this does is to first dilute the CFU’s in the urine into a larger volume of the broth, and then inhibit growth of the bacteria by refrigerating the enrichment broth. Further research is warranted to see if perhaps incubating the tryptic soy broth at warmer temperatures, after inoculation with the urine sample, can produce a higher sensitivity for UTI detection before we can really rule out the utility of this growth media in our urine sample storage.

Abbreviations:
CFU – Colony forming unit
TSB – Tryptic soy broth
UTI – Urinary tract infection

References:
1. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/TrypticSoyBroth.htm
2. https://www.bd.com/resource.aspx?IDX=30505
3. Acierno MJ, Partyka M, Waite K, et al. Effect of refrigeration of clinical canine urine samples on quantitative bacterial culture. JAVMA 2018;253(2):177-180.

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