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Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 261-264
Evaluation of commercial boric acid containing vials for urine culture: Low risk of contamination and cost effectiveness considerations

Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication24-Oct-2013


Background: Urine culture is a gold standard in the diagnosis of urinary tract infection. Clean catch midstream urine collection and prompt transportation is essential for appropriate diagnosis. Improper collection and delay in transportation leads to diagnostic dilemma. In developing countries, higher ambient temperatures further complicate the scenario. Here, we have evaluated the role of boric acid as a preservative for urine samples prior to culture in female patients attending outpatient department at our center. Materials and Method: Consecutive 104 urine samples were cultured simultaneously in plain uricol (Control-C) and boric acid containing tubes from Becton Dickinson urine culture kit (Boric acid group-BA). Results: In the real-time evaluation, we found that in almost 57% (59/104) of the urine samples tested, it was more effective in maintaining the number of the organisms as compared to samples in the container without any preservative. Our in vitro study of simulated urine cultures revealed that urine samples could be kept up to 12 h before culture in the preservative without any inhibitory effect of boric acid. Though the use of boric acid kit may marginally increase the initial cost but has indirect effects like preventing delays in treatment and avoidance of false prescription of antibiotics. If the man-hours spent on repeat investigations are also taken into consideration, then the economic cost borne by the laboratory would also decrease manifold with the use of these containers.

Keywords: Boric acid, preservative, urine culture

How to cite this article:
Appannanavar SB, Biswal M, Rajkumari N, Mohan B, Taneja N. Evaluation of commercial boric acid containing vials for urine culture: Low risk of contamination and cost effectiveness considerations . Indian J Pathol Microbiol 2013;56:261-4

How to cite this URL:
Appannanavar SB, Biswal M, Rajkumari N, Mohan B, Taneja N. Evaluation of commercial boric acid containing vials for urine culture: Low risk of contamination and cost effectiveness considerations . Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Jun 4];56:261-4. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/261/120386

   Introduction Top

Till today urine culture remains the gold standard for diagnosis of urinary tract infections. The diagnosis of urinary tract infection is complicated in certain patients because of the difficulty in obtaining clean catch midstream urine. This is especially true for female patients due to the presence of contaminating urethral, skin, genital and fecal flora. Overcrowding is a common scenario in developing countries like ours, where the time spent per patient is usually insufficient to explain proper collection of urine sample. Failure to collect clean mid-catch urine, results in either over reporting of non-significant number as significant or under reporting by missing a true pathogen due to contaminating organism from normal flora. In a month at our 1900 bedded tertiary care referral center, we face diagnostic dilemma in around 17% (91/547) of all the samples received from female patients due to mixed flora or gross contamination signifying improper collection. Further, in a semi-quantitative culture of voided urine, a count of more than 10 5 CFU/mL is considered significant. This would be appropriate only when the sample is processed within two hours or maintained at 4°C. However, this may not be possible as there is an inevitable delay in the transportation of sample to the routine microbiology laboratory. In a tropical country, this problem is compounded by a high ambient temperature which can reach between 35 and 40°C.

To circumvent this problem, chemicals like sodium chloride polyvinylpyrrolidone (NaCl PVP) and boric acid have been recommended as urine preservatives. Boric acid (BA) is a well-established preservative for urine. [1],[2] However, there is a cost constraint as boric acid containing containers are substantially more expensive than plain sterile containers for the collection of urine samples. So, we carried out an efficacy analysis of using Becton Dickinson urine culture kit (BD kit) containing boric acid versus plain containers in a cohort of female patients attending the outpatient department of our hospital. We also compared the toxic effect of boric acid on various organisms at different time intervals in higher ambient temperature which would be more appropriate for tropical countries like India. Finally, we evaluated the cost effectiveness of commercial boric acid containing vials routinely in this group of patients.

   Materials and Methods Top

The study was divided into two phases. In the first phase, a trained microbiology technical team supervised the collection of clean voided midstream urine from female patients with suspected UTI or bacteriuria who had been referred to the outpatient department for sample collection. This part was carried out in the months of June and July 2011. Prior to collection, patients were instructed to clean periurethral area using antiseptic swab provided with the Becton Dickinson urine culture kit (BD kit). Midstream urine was then collected in a sterile container provided with the kit. Immediately after collection, a part of the sample was transferred to the boric acid containing vacutainer tube present in the kit. Both containers were stored and transported under similar ambient temperatures to the routine microbiology laboratory for further processing. Urine samples in both containers were cultured simultaneously using a 1 μL loop onto Cysteine Lactose Electrolyte Deficient (CLED) media. After overnight incubation, the colony count from both containers was enumerated and recorded as colony forming unit per mL (CFU/mL).

In the second phase of study, we wanted to evaluate the toxic effect of boric acid at different time intervals in serial dilutions of the commonly isolated pathogens. Overnight broth cultures of uropathogens: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus spp. and Staphylococcus aureus were adjusted to 0.5 McFarland (1.5 × 10 8 CFU/mL). Each of the above organisms was spiked into sterile parallel set of plain tubes (non-BA) and boric acid vacutainers of BD kit (BA) containing known volumes of autoclaved urine to achieve a final count of 10 7 CFU/mL. Serial dilutions of the spiked urine were made to obtain concentrations from 10 5 to 10 2 CFU/mL of each organism per mL in both the sets of tubes (BA and non-BA). All the tubes were maintained at 32°C and serial cultures were performed on CLED using 1 μmL loop at 3, 5, 12, 24 and 48 hours. After overnight incubation, the colony count in each dilution was recorded and expressed as CFU/mL. The colony counts obtained in the plain tubes were considered as the control (non-BA group) number of organisms that the organism would achieve without any inhibitors. These counts were used to compare the counts obtained in BA containing tubes to assess a) the efficacy of BA as preservative and b) toxic effect on organisms at different time intervals.

   Results Top

Phase 1

In the real-time assessment of boric acid, a total of 104 voided urine samples from female patients were analyzed for the efficacy of boric acid as preservative in comparison to plain tubes. Seventeen samples (16.3%) were culture negative in both the groups. In the remaining 87 samples with culture positivity, no difference in growth was noted in 32.18% (28/87) of the samples.

Discrepancy was noted in the remaining 67.81% (59/87) samples showing culture positivity, 37.28% (22/59) showed some growth in non-BA group but were culture negative in BA group. Of these 22 samples, ten were from female patients who had submitted samples to rule out asymptomatic bacteriuria as part of antenatal check-up protocol, and of these 20% (2/10) had a count of >10 5 CFU/mL [Table 1]. Remaining twelve patients had complicated UTI, attending urology (n = 4) and radiotherapy OPD (n = 8) who had a count of >10 3 CFU/mL (cut-off for complicated UTI).

In remaining 37 samples (37/59 - 62.17%): 33 samples had higher CFU in non-BA group of which 72% (24/33) had less than 1 log 10 difference and 28% (9/33) had more than 1 log 10 difference in CFU/mL count. Among the 9 samples with >1 log 10 difference, 45% (4/9) had a non-significant bacteriuria count of 10 3 CFU/mL in BA while the non-BA tubes showed a count in the significant bacteriuria range (10 5- 10 8 CFU/mL). In the remaining four samples, higher CFU/mL was noted in BA tubes as compared to non-BA tubes. The logarithmic difference in growth of the most common isolates obtained with and without BA is shown in [Table 2].
Table 1: Total number of organisms and CFU/mL in control tubes but no growth in BA tubes

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Table 2: Table listing organism-wise diff erence in growth between BA and control groups

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Phase 2

In the phase 2 of the study, we compared the growth rate of all these organisms at different time intervals and at different initial dilutions in containers with and without BA following incubation at 32°C. As compared to BA, control group showed increased growth of organisms by > 1 log 10 CFU/mL after 5 h of incubation in all organisms except Enterococcus. The extent and statistically significant difference between the two groups are shown in [Figure 1]. We also noted the inhibitory effect of boric acid on the most commonly isolated organisms at different time intervals. No inhibitory effect of boric acid was noted for E. coli and Enterococcus spp. even after 48 h of incubation. A moderate inhibition was noted for K. pneumoniae and P. aeruginosa with a fall of 1 and 2 log 10 , respectively, as compared to initial dilution after 12 h of incubation. Inhibitory effect of BA was noted after 5 h of incubation for S. aureus and counts fell to <10 2 CFU/mL after 48 h of incubation.
Figure 1: In vitro study showing the diff erence in growths (CFU/mL expressed as log10) of common uropathogens in Boric Acid (BA) and non-BA (C) groups

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Comparative cost analysis

We estimate that the financial cost of each urine culture is Rs 110, including the price of sterile container, culture media, biochemicals and antibiotic susceptibility testing. For each report of "overgrowth of normal flora" or "mixed bacterial growth of unknown significance", a repeat culture after proper precaution was advised. In a month, around 28.33% (155/547) urine samples sent for culture sensitivity were repeated in our laboratory due to doubtful significance or gross contamination. Nearly 60% (91/155) of these samples were from female patients. If we calculate the cost for single culture using BD kit (Rs 140) and double cultures for repeat samples (28%) using conventional method, the difference was only marginal.

   Discussion Top

The overcrowded outpatient departments in a developing country see a lot of uneducated patients who have poor hygienic conditions. At our center, we receive more than 30-40 thousand urine samples annually for culture and sensitivity. The collection area is centralized and receives >1000 microbiology samples each day. The sample collection area and refrigeration facility both do not have the capacity to deal with such high loads. Urine samples are collected in sterile containers and there are unavoidable logistic issues for transportation under refrigerated conditions for about 100 urine samples to be transported in cold chain. Due to overcrowding it becomes very difficult to explain to each patient, the right way of collecting clean catch midstream urine. The problem is more so in female patients in whom sample collection is difficult due to anatomical limitations.

In a year, we have noted that nearly 30% of urine samples are received from female patients attending antenatal clinic, urology OPD or radiotherapy OPD. Female patients face difficulties in collecting clean catch midstream urine without contamination from commensal flora. In our laboratory, we face diagnostic dilemma in up to 17% of the urine samples received from this group of patients. This is due to inevitable delay in sample processing, which results in overgrowth of contaminating flora. In our setting, complicated UTI is very common and hence a repeat culture is requested in case of overgrowth of normal flora. When we evaluated the efficacy of boric acid as a preservative for urine samples and compared it with our routine laboratory system, we noted that in 57% (59/104) of the samples, there was a discrepancy between boric acid and non-boric acid group.

Optimum sample collection is very essential in a semi-quantitative culture, where colony count is very critical. A count of ≥10 5 CFU/mL is considered significant assuming that a clean catch midstream urine is transported and plated within 1-2 h. Urine being a good culture medium if left standing after collection can lead to multiplication of normal flora and misreporting due to missing of true pathogen masked by overgrown flora. Hindman et al. have shown that delays of even greater than 2 h in inoculating cultures may produce results which could cause errors in diagnosis. [3] In a source-limited setup like ours, a minimum of three to three and half hour delay is generally expected. Refrigeration may be a good alternative but may not be possible due to higher volume of samples received each day. Two chemicals namely NaCl polyvinylpyrrolidone (PVP) and boric acid have been used previously to eliminate the need for refrigeration of urine samples. [4] The activity of NaCl PVP is based on the bacteriostatic activity of NaCl, and PVP serves as a protective colloid and helps in dispersal of bacteria. In clinical trials, although NaCl PVP was found to be efficacious, but decline in bacterial growth was noted over 6-8 h. [4] Boric acid when used at a concentration of 1.8% has shown to be effective for up to 4 days. [5]

In previous studies, BD urine culture kit containing boric acid was shown to be as effective as refrigeration in maintaining urine reliably for at least 24 h before quantitative culture [1],[6] However, the evaluation was done at lower ambient temperatures which may not be extrapolated to our unique set-up having higher temperatures that can reach up to 35-40°C. [1],[6] Moreover, in these studies, efficacy of boric acid as a preservative was compared with refrigeration of urine sample. Result of phase one experiment from our study will be more appropriate for developing countries as it was conducted at a higher ambient temperature and compared in real time with routine laboratory system. In tropical countries, overcrowding and high ambient temperatures further complicate the problem. In an overcrowded setting like the OPD sample collection center, it is not possible to ensure that every patient collects sample observing the recommended precautions. It is therefore imperative that the process of sample collection be simplified. Additional advantage of the Becton Dickinson kit includes a swab for cleaning the external urethral meatus as well as a boric acid containing vacutainer, which makes the process much more convenient for the patient.

We observed that 38% of samples (22/59) had a growth of up to 10 3- 10 6 in non-BA group and 63% (14/22) of these samples were having significant bacteriuria with a count of ≥10 3 CFU/mL in non-BA group, while no growth in BA group. Majority of these patients were having complicated UTI who had either anatomically or physiologically compromised urinary tract where a count of 10 3 CFU/mL will be considered significant. This can lead to unnecessary prescription of the antibiotics and its attendant problems like selecting resistant strains. This will also increase the burden on the laboratory that has to cater for repeat cultures.

The limitation of the first phase of the study was that the original number of organisms in the sample was not determined. Thus in the second phase of the study, we tried to simulate the delays by inoculating at staggered intervals with known initial dilution of spiked organism. The bacterial count of the preserved specimen was maintained at a constant level till 24 and 48 h in E. coli and Enterococcus, respectively. However, inhibition was noted for K. pneumoniae, P. aeruginosa and S. aureus after 12 h. Porter et al. observed no inhibitory effect of boric acid on E. coli but viable counts of some strains of Proteus spp. and Klebsiella spp. were found to be reduced in their study. [5] In a comparative study, Watson et al. found that boric acid was toxic for Pseudomonas in less than 24 h and toxic for E. coli and Proteus spp. after 24 h. [7] In contrast Guenther et al. found boric acid to be as effective as refrigeration in maintaining urine for up to 24 h before quantitative culture. [6] In their study, they showed that if the transit time in the kit does not exceed 24 h, and 10 4 CFU per mL in preserved urine is regarded as a positive result, agreement between results for cultures of original urine specimens yielding >10 5 CFU per mL, and results for preserved urine specimens is within acceptable limits. [6] These results may not be appropriate for our set-up, where we deal with complicated UTIs in whom a cutoff of 10 3 CFU/mL or below is considered significant. Moreover, we noted that the rate of growth of organisms in boric acid was maintained only up to 12 h for majority of the organism tested except E. coli and Enterococcus. Thus, we recommend that boric acid as a preservative may be kept up to 12 h in our set-up.

Use of boric acid kit may marginally increase the initial cost but has indirect effects like preventing delays in treatment and avoidance of false prescription of antibiotics. If the man-hours spent on repeat investigations are also taken into consideration, then the economic cost borne by the laboratory would also decrease manifold with the use of these containers. Thus we recommend that BD kit is useful in females attending the outpatient sections in whom it is not only cost effective but also convenient to use.

   References Top

1.Lauer BA, Reller LB, Mirrett S. Evaluation of preservative fluid for urine collected for culture. J Clin Microbiol 1979;10:42-5.  Back to cited text no. 1
2.Meers PD, Chow CK. Bacteriostatic and bactericidal actions of boric acid against bacteria and fungi commonly found in urine. J Clin Pathol 1990;43:484-7.  Back to cited text no. 2
3.Hindman R, Tronic B, Bartlett R. Effect of delay on culture of urine. J Clin Microbiol 1976;4:102-3.  Back to cited text no. 3
4.Amies CR, Corpas A. A preservative for urine specimens in transit to the bacteriological laboratory. J Med Microbiol 1971;4:362-5.  Back to cited text no. 4
5.Porter IA, Brodie J. Boric acid preservation of urine samples. Br Med J 1969;2:353-5.  Back to cited text no. 5
6.Guenther KL, Washington JA 2nd. Evaluation of the B-D Urine Culture Kit. J Clin Microbiol 1981;14:628-30.  Back to cited text no. 6
7.Watson PG, Duerden BI. Laboratory assessment of physical and chemical methods of preserving urine specimens. J Clin Pathol 1977;30:532-6.  Back to cited text no. 7

Correspondence Address:
Neelam Taneja
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.120386

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